Oral gavage of capsaicin causes TRPV1-dependent acute hypothermia and TRPV1-independent long-lasting increase of locomotor activity in the mouse.

Capsaicin (CAP), the pungent ingredient of hot red pepper, is a selective ligand for the heat-sensitive transient receptor potential V1 cation channel 1 (TRPV1). Although CAP has been traditionally used as the ingredient of spices for various foods in the world, the effect of oral intake of CAP on thermoregulation and locomotor activity, and CAP-induced activation of brain neural circuits are not well understood. In this study, therefore, we examined the effects of oral gavage of CAP on core body and tail surface temperature, locomotor activity, and Fos expression in thermoregulation- and sensory information-associated hypothalamic and medullary brain regions using freely moving mice. Oral gavage of CAP acutely decreased core body temperature and alternatively increased tail surface temperature of wild type (WT) mice, whereas such acute temperature changes were not observed in TRPV1 knockout (KO) animals. Moreover, a long-lasting increase of locomotor activity was observed in both WT and TRPV1 KO mice after oral gavage of CAP, but increase in core body temperature was seen only in TRPV1 KO animals. Oral gavage of CAP induced neuronal Fos expression in the circumventricular organs, median and medial preoptic area, arcuate nucleus, and nucleus of the solitary tract, whereas neuronal Fos expression was scarcely observed in TRPV1 KO mice. Thus, the present study demonstrates in the mice that oral intake of CAP causes TRPV1-dependent acute hypothermia and TRPV1-independent long-lasting increase of locomotor activity, and moreover activates the brain circuits controlling thermoregulation and metabolism.

Structural Reconstruction of the Perivascular Space in the Adult Mouse Neurohypophysis During an Osmotic Stimulation.

Oxytocin (OXT) and arginine vasopressin (AVP) neuropeptides in the neurohypophysis (NH) control lactation and body fluid homeostasis, respectively. Hypothalamic neurosecretory neurones project their axons from the supraoptic and paraventricular nuclei to the NH to make contact with the vascular surface and release OXT and AVP. The neurohypophysial vascular structure is unique because it has a wide perivascular space between the inner and outer basement membranes. However, the significance of this unique vascular structure remains unclear; therefore, we aimed to determine the functional significance of the perivascular space and its activity-dependent changes during salt loading in adult mice. The results obtained revealed that pericytes were the main resident cells and defined the profile of the perivascular space. Moreover, pericytes sometimes extended their cellular processes or 'perivascular protrusions' into neurohypophysial parenchyma between axonal terminals. The vascular permeability of low-molecular-weight (LMW) molecules was higher at perivascular protrusions than at the smooth vascular surface. Axonal terminals containing OXT and AVP were more likely to localise at perivascular protrusions than at the smooth vascular surface. Chronic salt loading with 2% NaCl significantly induced prominent changes in the shape of pericytes and also increased the number of perivascular protrusions and the surface area of the perivascular space together with elevations in the vascular permeability of LMW molecules. Collectively, these results indicate that the perivascular space of the NH acts as the main diffusion route for OXT and AVP and, in addition, changes in the shape of pericytes and perivascular reconstruction occur in response to an increased demand for neuropeptide release.

Periodontal tissue engineering by nano beta-tricalcium phosphate scaffold and fibroblast growth factor-2 in one-wall infrabony defects of dogs.

BACKGROUND AND OBJECTIVE: Nanoparticle bioceramics are being investigated for biomedical applications. We fabricated a regenerative scaffold comprising type I collagen and beta-tricalcium phosphate (ß-TCP) nanoparticles. Fibroblast growth factor-2 (FGF-2) is a bioeffective signaling molecule that stimulates cell proliferation and wound healing. This study examined the effects, on bioactivity, of a nano-ß-TCP/collagen scaffold loaded with FGF-2, particularly on periodontal tissue wound healing. MATERIAL AND METHODS: Beta-tricalcium phosphate was pulverized into nanosize particles (84 nm) and was then dispersed. A nano-ß-TCP scaffold was prepared by coating the surface of a collagen scaffold with a nanosize ß-TCP dispersion. Scaffolds were characterized using scanning electron microscopy, compressive testing, cell seeding and rat subcutaneous implant testing. Then, nano-ß-TCP scaffold, nano-ß-TCP scaffold loaded with FGF-2 and noncoated collagen scaffold were implanted into a dog one-wall infrabony defect model. Histological observations were made at 10 d and 4 wk postsurgery. RESULTS: Scanning electron microscopy images show that TCP nanoparticles were attached to collagen fibers. The nano-ß-TCP scaffold showed higher compressive strength and cytocompatibility compared with the noncoated collagen scaffold. Rat subcutaneous implant tests showed that the DNA contents of infiltrating cells in the nano-ß-TCP scaffold and the FGF-2-loaded scaffold were approximately 2.8-fold and 3.7-fold greater, respectively, than in the collagen scaffold. Histological samples from the periodontal defect model showed about five-fold greater periodontal tissue repair following implantation of the nano-ß-TCP scaffold loaded with FGF-2 compared with the collagen scaffold. CONCLUSION: The ß-TCP nanoparticle coating strongly improved the collagen scaffold bioactivity. Nano-ß-TCP scaffolds containing FGF-2 are anticipated for use in periodontal tissue engineering.

Activity-dependent Notch signalling in the hypothalamic-neurohypophysial system of adult mouse brains.

Notch signalling has a key role in cell fate specification in developing brains; however, recent studies have shown that Notch signalling also participates in the regulation of synaptic plasticity in adult brains. In the present study, we examined the expression of Notch3 and Delta-like ligand 4 (DLL4) in the hypothalamic-neurohypophysial system (HNS) of the adult mouse. The expression of DLL4 was higher in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) compared to adjacent hypothalamic regions. Double-labelling immunohistochemistry using vesicular GABA transporter and glutamate transporter revealed that DLL4 was localised at a subpopulation of excitatory and inhibitory axonal boutons against somatodendrites of arginine vasopressin (AVP)- and oxytocin (OXT)-containing magnocellular neurones. In the neurohypophysis (NH), the expression of DLL4 was seen at OXT- but not AVP-containing axonal terminals. The expression of Notch3 was seen at somatodendrites of AVP- and OXT-containing magnocellular neurones in the SON and PVN and at pituicytes in the NH. Chronic physiological stimulation by salt loading, which remarkably enhances the release of AVP and OXT, decreased the number of DLL4-immunoreactive axonal boutons in the SON and PVN. Moreover, chronic and acute osmotic stimulation promoted proteolytic cleavage of Notch3 to yield the intracellular fragments of Notch3 in the HNS. Thus, the present study demonstrates activity-dependent reduction of DLL4 expression and proteolytic cleavage of Notch3 in the HNS, suggesting that Notch signalling possibly participates in synaptic interaction in the hypothalamic nuclei and neuroglial interaction in the NH.

Kidney fibrosis is independent of the amount of ascorbic acid in mice with unilateral ureteral obstruction.

In response to sustained damage to a kidney, fibrosis that can be characterized as the deposition of a collagenous matrix occurs and consequently causes chronic kidney failure. Because most animals used in experiments synthesize ascorbic acid (AsA) from glucose, the roles of AsA in fibrotic kidney diseases are largely unknown. Unilateral ureteric obstruction (UUO) mimics the complex pathophysiology of chronic obstructive nephropathy and is an ideal model for the investigation of the roles of AsA in kidney failure. We examined the impact of a deficiency of Akr1a, a gene that encodes aldehyde reductase and is responsible for the production of AsA, on fibrotic damage caused by UUO in mice. Oxidatively modified DNA was elevated in wild-type and Akr1a-deficient kidneys as a result of UUO to a similar extent, and was only slightly suppressed by the administration of AsA. Even though Akrla-deficient mice could produce only about 10% of the AsA produced by wild-type mice, no difference was observed in collagen I synthesis under pathological conditions. The data implied either a low demand for AsA or the presence of another electron donor for collagen I production in the mouse kidney. Next, we attempted to elucidate the potential causes for oxidative damage in kidney cells during the fibrotic change. We found decreases in mitochondrial proteins, particularly in electron transport complexes, at the initial stage of the kidney fibrosis. The data imply that a dysfunction of the mitochondria leads to an elevation of ROS, which results in kidney fibrosis by stimulating cellular transformation to myofibroblasts.

Effective cell collection method using collagenase and ultrasonic vibration.

This study proposes a novel cell collection method based on collagenase treatment and ultrasonic vibration. The method collects calf chondrocytes from a reusable metal cell culture substrate. To develop our concept, we calculated the natural vibration modes of the cell culture substrate by a finite element method, and conducted eigenvalue and piezoelectric-structural analyses. Selecting the first out-of-plane vibration mode of the substrate, which has a single nodal circle, we designed and fabricated the cell collection device. The excited vibration mode properly realized our intentions. We then evaluated the cell collection ratio and the growth response, and observed the morphology of the collected cells. The collagenase and ultrasonic vibration treatment collected comparable numbers of cells to conventional trypsin and pipetting treatment, but improved the proliferating cell statistics. Morphological observations revealed that the membranes of cells collected by the proposed method remain intact; consequently, the cells are larger and rougher than cells collected by the conventional method. Therefore, we present a promising cell collection method for adhesive cell culturing process.

Separation method of blood constituents using dielectrophoresis and flow-induced shear force.

Platelet Rich Plasma (PRP) contains many cytokines for treatment skin diseases. The final goal of this study is to develop the dielectrophoretic PRP purification system enriching the platelets in a label-free manner from whole blood solution. In this study, we characterized dielectrophoretic properties of red blood cell, white blood cell, and platelet for the fundamental study. Moreover, purification of PRP was performed to eliminate red and white blood cells using dielectrophoretic and flow-induced shear force.

High throughput cell sorting device using dielectrophoresis and fluid-induced shear force.

Cell sorting is important technology in many areas such as medical diagnosis in hospitals and cell engineering. Dielectrophoresis (DEP) is one of the promising approaches for cell separation because it does not require any fluorescent dye or antibody. In our previous study, we developed DEP cells sorting device, however the amount of throughput was not sufficient. In this study, we developed high throughput cell sorting device using dielectrophoretic and fluid-induced shear force. As the pilot study, mixed solution containing bovine chondrocytes and polystyrene beads were precisely separated using our novel DEP cell sorting device.

VEGF-dependent continuous angiogenesis in the median eminence of adult mice.

Brain vasculature forms the blood-brain barrier (BBB) that restricts the movement of molecules between the brain and blood, but the capillary of the median eminence (ME) lacks the BBB for secretion of adenohypophysial hormone-releasing peptides. In the present study, we aimed to elucidate whether continuous angiogenesis occurs in the ME of adult mice. By using a mitotic marker, bromodeoxyuridine (BrdU), we demonstrated that new endothelial cells were born continuously in the ME of adults. Prominent expression of NG2, platelet-derived growth factor receptor B (PDGFRB), and delta-like ligand 4 was observed at pericytes of adults, although the expression of these angiogenesis-associated proteins has been shown to be at low or trace levels in adult mature capillary. In addition, vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, was expressed highly in the nervous parenchyma of the ME. Expression of VEGF receptor 2 (VEGFR2) was observed at endothelial cells in the external zone and at somatodendrites in the internal zone. Finally, a VEGFR- and PDGFR-associated tyrosine kinase inhibitor, SU11248, significantly decreased the number of BrdU-positive proliferating endothelial cells and parenchyma cells. In conclusion, the present study demonstrates VEGF-dependent continuous angiogenesis in the ME of adult mouse brains under normal conditions, which provides new insight into our understanding of neurosecretion in the ME.

Oxygen reduction activity of carbon nitride supported on carbon nanotubes.

Fuel cells offer an alternative to burning fossil fuels, but use platinum as a catalyst which is expensive and scarce. Cheap, alternative catalysts could enable fuel cells to become serious contenders in the green energy sector. One promising class of catalyst for electrochemical oxygen reduction is iron-containing, nanostructured, nitrogen-doped carbon. The catalytic activity of such N-doped carbons has improved vastly over the years bringing industrial applications ever closer. Stoichiometric carbon nitride powder has only been observed in recent years. It has nitrogen content up to 57% and as such is an extremely interesting material to work with. The electrochemical activity of carbon nitride has already been explored, confirming that iron is not a necessary ingredient for 4-electron oxygen reduction. Here, we synthesize carbon nitride on a carbon nanotube support and subject it to high temperature treatment in an effort to increase the surface area and conductivity. The results lend insight into the mechanism of oxygen reduction and show the potential for carbon nanotube-supported carbon nitride to be used as a catalyst to replace platinum in fuel cells.

Transient expression of Xpn, an XLMR protein related to neurite extension, during brain development and participation in neurite outgrowth.

KIAA2022 has been implicated as a gene responsible for expressing X-linked mental retardation (XLMR) proteins in humans. However, the functional role of KIAA2022 in the human brain remains unclear. Here, we revealed that depletion of Kiaa2022 inhibits neurite outgrowth of PC12 cells, indicating that the gene participates in neurite extension. Thus, we termed Kiaa2022 as an XLMR protein related to neurite extension (Xpn). Using the mouse brain as a model and ontogenetic analysis of Xpn by real-time PCR, we clearly demonstrated that Xpn is expressed transiently during the late embryonic and perinatal stages. In situ hybridization histochemistry further revealed that Xpn-expressing neurons could be categorized ontogenetically into three types. The first type showed transient expression of Xpn during development. The second type maximally expressed Xpn during the late embryonic or perinatal stage. Thereafter, Xpn expression in this type of neuron decreased gradually throughout development. Nevertheless, a significant level of Xpn expression was detected even into adulthood. The third type of neurons initiated expression of Xpn during the embryonic stage, and continued to express the gene throughout the remaining developmental stages. Subsequent immunohistochemical analysis revealed that Xpn was localized to the nucleus and cytoplasm throughout brain development. Our findings indicate that Xpn may participate in neural circuit formation during developmental stages via nuclear and cytoplasmic Xpn. Moreover, disturbances of this neuronal circuit formation may play a role in the pathogenesis of mental retardation.

Learning curve of laparoscopy-assisted gastrectomy using a standardized surgical technique and an established educational system.

BACKGROUND AND AIMS: The learning curve of a trainee for laparoscopy-assisted gastrectomy in a high volume center, in which an educational system and a standardized laparoscopic procedure are already established, remains unclear. MATERIAL AND METHODS: The early surgical outcomes of the patients of two trainees were investigated. Both trainees followed a training program where they performed at least 20 cases being the camera assistant, 20 cases being the first assistant, before performing the surgery as an operator. RESULTS: The average operation time, intraoperative bleeding, the number of retrieved lymph nodes, and morbidity rate were 240.2 min, 45.7 ml, 35.4, and 13.0%, respectively. There was no learning curve effect observed except with the operation time of one trainee. CONCLUSIONS: In a high volume center with an established educational system, trainees could perform laparoscopy-assisted gastrectomy safely, although there might be a -learning curve effect in operation time and the surgeries took longer operation time in trainee's initial cases.

Monthly administration of a novel PTH-collagen binding domain fusion protein is anabolic in mice.

We synthesized fusion proteins of parathyroid hormone (PTH) (1-33) and the collagen binding domain of ColH (CBD) and tested them for anabolic bone activity in mice. Two fusion proteins were synthesized, linking the carboxy terminus of PTH(1-33) either directly to the amino terminal of the CBD or to the CBD through an adjacent ColH domain (PTH-PKD-CBD). Both PTH-CBD and PTH-PKD-CBD increased cAMP accumulation in cells stably transfected with the PTH/PTHrP receptor, and both peptides bound to type 1 collagen in flow-through assays. Distribution studies indicated that the PTH-CBD was concentrated in the bone and skin, tissues with abundant collagen and blood flow. Administration of 320 µg/kg PTH-CBD either weekly (for 8 weeks) or monthly (for 6 months) to 7-week-old C57BL/6J mice resulted in a sustained increase in bone mineral density (BMD) (15% for weekly studies, 13% for monthly studies; P < 0.05). PTH-PKD-CBD showed only 5% increases in BMD after weekly administration, and, as expected, neither weekly nor monthly PTH(1-34) affected BMD. PTH-CBD increased serum alkaline phosphatase levels. Importantly, there were no significant increases in serum calcium observed. Collectively, the data suggest that PTH-CBD has a sustained anabolic effect in bone with either weekly or monthly administration. This approach of targeted delivery of PTH to bone may show promise for the treatment of disorders of low bone mass, such as postmenopausal osteoporosis.

Mixture of cis-3-hexenol and trans-2-hexenal attenuates behavioral and stress responses induced by 2,5-dihydro-2,4,5-trimethylthiazoline and electric footshock stress in rats.

Green odor (GO), a mixture of cis-3-hexenol and trans-2-hexenal, attenuates stress responses and anxiety to psychological stressors in rodents; however, it remains unknown whether GO affects behavioral and stress responses to risk-related olfactory stimuli and actual noxious stimuli. The present study investigated the effects of green odor on behavioral and plasma adrenocorticotropic hormone (ACTH) responses to 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), a component of fox feces, and electric footshock (FS) stress. When rats were simultaneously exposed to TMT and GO, they showed decreases in immobility and plasma ACTH levels compared with TMT alone. GO exposure after TMT increased immobility, but blocked the elevation of plasma ACTH levels compared with rats exposed to distilled water after TMT. This means that GO presentation during TMT attenuated the TMT-induced behavioral response and GO presentation during and after TMT inhibited TMT-induced elevation of plasma ACTH levels. Furthermore, electric FS-induced plasma ACTH elevations were attenuated by simultaneous GO and FS exposure. GO presentation after FS attenuated plasma ACTH elevations and fecal responses. These findings reveal that GO has alleviating effects on olfactory stimulus- and noxious stimulus-induced behavioral and endocrinal responses.

Activity-dependent remodeling of chondroitin sulfate proteoglycans extracellular matrix in the hypothalamo-neurohypophysial system.

The hypothalamo-neurohypophysial system (HNS) consisting of arginine vasopressin (AVP) and oxytocin (OXT) magnocellular neurons shows the structural plasticity including the rearrangement of synapses, dendrites, and neurovascular contacts during chronic physiological stimulation. In this study, we examined the remodeling of chondroitin sulfate proteoglycans (CSPGs), main extracellular matrix (ECM), in the HNS after salt loading known as a chronic stimulation to cause the structural plasticity. In the supraoptic nucleus (SON), confocal microscopic observation revealed that the immunoreactivity of 6B4 proteoglycans (PG) was observed mainly at AVP-positive magnocellular neurons but that of neurocan was seen chiefly at OXT-positive magnocellular neurons. The immunoreactivity of phosphacan and aggrecan was seen at both AVP- and OXT-positive magnocellular neurons. Electron microscopic observation further showed that the immunoreactivity of phosphacan and neurocan was observed at astrocytic processes to surround somata, dendrites, and terminals, but not synaptic junctions. In the neurohypophysis (NH), the immunoreactivity of phosphacan, 6B4 PGs, and neurocan was observed at AVP-positive magnocellular terminals, but the reactivity of Wisteria floribunda agglutinin lectin was seen at OXT-positive ones. The immunoreactivity of versican was found at microvessel and that of aggrecan was not detected in the NH. Quantitative morphometrical analysis showed that the chronic physiological stimulation by 7-day salt loading decreased the level of 6B4 PGs in the SON and the level of phosphacan, 6B4 PGs, and neurocan in the NH. These results suggest that the extracellular microenvironment of CSPGs is different between AVP and OXT magnocellular neurons and activity-dependent remodeling of CSPGs could be involved in the structural plasticity of the HNS.

Functional difference of receptor-type protein tyrosine phosphatase zeta/beta isoforms in neurogenesis of hippocampal neurons.

Receptor-type protein tyrosine phosphatase zeta/beta (RPTPzeta) is a transmembrane chondroitin sulfate proteoglycan (CSPG) and has been shown to play crucial roles in controlling axonal growth and neuronal migration. The RPTPzeta has two transmembranous isoforms, shorter receptor form of RPTPzeta (sRPTPzeta) and full-length receptor form of RPTPzeta (fRPTPzeta), but no studies have been reported about functional difference of these two isoforms. In the present study, therefore, we examined whether or not two RPTPzeta isoforms have different role in controlling dendritic morphology and synaptic number in cultured hippocampal neurons using the quantitative morphometrical analysis. Confocal microscopic observation showed that the immunoreactivity of RPTPzeta was observed throughout cells such as axons, growth cones, and dendrites at the early stages of neuronal culture, while it was seen predominantly on dendrites at the late stages. Western blotting analysis revealed that fRPTPzeta was mainly expressed at the early stages of culture and both RPTPzeta isoforms were expressed at late stages of culture. The overexpression of sRPTPzeta in hippocampal neurons increased the dendritic arborization without altering the average length of dendritic branches, whereas that of fRPTPzeta decreased the dendritic arborization and increased the average length of dendritic branches. The RNA interference of fRPTPzeta expression increased the dendritic arborization without altering the average length of dendritic branches. The overexpression of fRPTPzeta decreased the density of hippocampal dendritic synapses, but that of sRPTPzeta had no effects. Pleiotrophin, a ligand for RPTPzeta to interfere the phosphatase activity, increased the density of hippocampal dendritic synapses. Thus, the present study demonstrates that two transmembranous RPTPzeta isoforms have different functions for regulating dendritogenesis and synaptogenesis.

The distribution and characterization of endogenous protein arginine N-methyltransferase 8 in mouse CNS.

Protein arginine N-methyltransferase (PRMT) 8 was first discovered from a database search for genes harboring four conserved methyltransferase motifs, which shares more than 80% homology to PRMT1 in amino acid [Lee J, Sayegh J, Daniel J, Clarke S, Bedford MT (2005) PRMT8, a new membrane-bound tissue-specific member of the protein arginine methyltransferase family. J Biol Chem 280:32890-32896]. Interestingly, its tissue distribution is strikingly restricted to mouse CNS. To characterize the function in the CNS neurons, we raised an antiserum against PRMT8 to perform immunohistochemistry (IHC) and Western blot analysis. By IHC, the immunoreactivity of endogenous PRMT8 was broadly distributed in the CNS neurons with markedly intense signals in the cerebellum, hippocampal formation, and cortex, but was not detected in the cerebellar granular layer. In some subset of the neurons, the immunoreactivity was observed in the dendrites and axon bundles. The subcellular localization of the immunoreactivity was dominantly nuclear, arguing against the original report that exogenously expressed PRMT8 localizes to the plasma membrane via the N-terminal myristoylation. A series of the exogenously expressed proteins with different in-frame translation initiation codons was tested for comparison with the endogenous protein in molecular size. The third initiator codon produced the protein that was equivalent in size to the endogenous and showed a similar localizing pattern in PC12 cells. In conclusion, PRMT8 is a neuron-specific nuclear enzyme and the N-terminus does not contain the glycine end for myristoylation target.

Consecutive national surveys of ABO-incompatible blood transfusion in Japan.

BACKGROUND AND OBJECTIVES: Morbidity and mortality from ABO-incompatible transfusion persist as consequences of human error. Even so, insufficient attention has been given to improving transfusion safety within the hospital. MATERIALS AND METHODS: National surveys of ABO-incompatible blood transfusions were conducted by the Japanese Society of Blood Transfusion, with support from the Ministry of Health, Labor and Welfare. Surveys concluded in 2000 and 2005 analysed ABO-incompatible transfusion data from the previous 5 years (January 1995 to December 1999 and January 2000 to December 2004, respectively). The first survey targeted 777 hospitals and the second, 1355 hospitals. Data were collected through anonymous questionnaires. RESULTS: The first survey achieved a 77.4% response rate (578 of 777 hospitals). The second survey collected data from 251 more hospitals, but with a lower response rate (61.2%, or 829 of 1355 hospitals). The first survey analysed 166 incidents from 578 hospitals, vs. 60 incidents from 829 hospitals in the second survey. The main cause of ABO-incompatible transfusion was identification error between patient and blood product: 55% (91 of 166) in the first survey and 45% (27 of 60) in the second. Patient outcomes included nine preventable deaths from 1995 to 1999, and eight preventable deaths from 2000 to 2004. CONCLUSION: Misidentification at the bedside persists as the main cause of ABO-incompatible transfusion.