Biomedical subjects

I Tooyama

Publications and source records attributed to I Tooyama.

At least 19 recordsLinked to original sources

PubMed2006

Comparison of MR images and histochemical localization of intra-arterially administered microglia surrounding beta-amyloid deposits in the rat brain.

The therapeutic use of microglial cells has recently received some attention for the treatment of Alzheimer disease (AD), but few non-invasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technique for tracking microglia injected intra-arterially in vivo. We micro-injected Abeta42 into the left hippocampus and saline into the right hippocampus of rats. We then administered microglia, which were labeled with enhanced green fluorescent protein (EGFP) gene and Resovist, into the carotid artery. After monitoring exogenously administered microglia using MRI, we compared the MR images and the histochemical localization of administered microglia. MRI revealed clear signal changes attributable to Resovist-containing microglia in Abeta-injected areas. Histochemistry demonstrated that EGFP-positive microglia accumulated around Abeta deposits and internalized the peptide. This study demonstrates the usefulness of MRI for non-invasive monitoring of exogenous microglia, and suggests a promising future for microglia/macrophages as therapeutic tools for AD.

Amyloid beta-Peptides↗

PubMed2005

Immunohistochemical study of TAFII250 in the rat laryngeal nervous system.

The cause of spasmodic dysphonia, a dystonic disorder of the larynx, remains unclear. Recently, TAFII250, TATA-box binding protein associated factor, was suggested to be involved in dystonia parkinsonism. There is a possibility that TAFII250 is involved in spasmodic dysphonia, but little information is available about the expression of TAFII250 in the laryngeal nervous system. In this study, we investigated the localization of TAFII250 protein in the rat laryngeal nervous system by immunohistochemistry. TAFII250-immunoreactivity was detected in the nodose ganglion and superior cervical ganglion. In these nuclei, TAFII250 was localized in the nucleus of NeuroTrace-positive neurons but not in GFAP-positive glial cells. No positive cells were detected in the motor and parasympathetic nervous system. TAFII250-immunoreactivity was sustained between 3 and 7 days after vagotomy, but at 14 days expression was down-regulated in the distal part of the nodose ganglion. These findings suggest that TAFII250 plays an important role in the laryngeal innervation of the sensory and sympathetic nervous systems.

Animals↗

PubMed2003

Expression of a splice variant of choline acetyltransferase in magnocellular neurons of the tuberomammillary nucleus of rat.

A splice variant of choline acetyltransferase mRNA has recently been identified in the pterygopalatine ganglion of rat. An antibody against this variant protein (designated pChAT) was demonstrated to immunolabel peripheral cholinergic neurons. In the present study, we investigated the expression of pChAT in rat brain. Amongst the brain regions examined, magnocellular neurons in the tuberomammillary nucleus of the posterior hypothalamus were immunohistochemically labelled with anti-pChAT antibody, whilst no immunolabelling was detected in cholinergic neurons in the basal forebrain or striatum. RT-PCR analysis confirmed the expression of pChAT mRNA in the posterior hypothalamus. The distribution of pChAT-positive neurons in the tuberomammillary nucleus was compared with that of neurons positive for adenosine deaminase, which is contained in all neurons of this nucleus. After colchicine treatment to inhibit axonal transport of enzyme, virtually all pChAT-positive cells contained adenosine deaminase. Conversely, about 85% of adenosine deaminase-positive cells contained pChAT in the ventral area, whilst 19% of adenosine deaminase-positive cells were pChAT-positive in the dorsal area. Long axonal projections of pChAT-positive cells in the tuberomammillary nucleus were shown by retrograde labelling of these cells after injection of cholera-toxin B subunit into the cerebral cortex. This study demonstrates that a splice variant of choline acetyltransferase is expressed in the tuberomammillary nucleus of rat. The results raise the possibility that some of the known diverse projection areas of this nucleus may have a cholinergic component.

Acetylcholine↗

PubMed2001

Acidic fibroblast growth factor (FGF-1) in the anterior horn cells of ALS and control cases.

The expression and localization of acidic fibroblast growth factor (aFGF; FGF-1) were examined in the spinal cord of patients with amyotrophic lateral sclerosis (ALS) and controls by reverse transcription-polymerase chain reaction (RT-PCR) method and immunohistochemistry. The RT-PCR experiments demonstrated that aFGF amplification products were clearly detected in all control cases but could be scarcely seen in ALS patients. aFGF immunoreactivity was detected in the anterior horn cells of the spinal cord. Double immunostaining for aFGF and choline acetyltransferase revealed that the majority (95.9%) of cholinergic neurons expressed aFGF. In ALS cases, the number and the staining intensity of aFGF-positive neurons were markedly decreased. These results suggest that aFGF is involved in ALS pathology.

Acetylcholine↗

PubMed2001

2-buten-4-olide, an endogenous feeding suppressant, improves spatial performance through brain acidic fibroblast growth factor in mice.

Endogenous sugar acid 2-buten-4-olide, a satiety substance, has been shown to increase the blood glucose, norepinephrine, and glucocorticoid concentrations that are known to modulate learning and memory processes. The glucose-induced release of acidic fibroblast growth factor facilitated the hippocampus-dependent memory function. In the present study, we investigated the effect of 2-buten-4-olide on the spatial performance of male DDY mice undergoing the water maze task. The intraperitoneal injection of 2-buten-4-olide (5 mg/kg) facilitated the spatial performance, which was indicated by a reduction in the escape latency in which the mouse finds and climbs the goal platform in comparison to the vehicle-injected control mice. In the probe test after removing the platform, the 2-buten-4-olide-treated mice stayed a longer time in the quadrant where the platform was originally located and crossed more frequently at the platform location than did the control mice. The pretreatment of acidic fibroblast growth factor antibody injected into the lateral ventricle eliminated the effect of 2-buten-4-olide both during the training sessions and during the probe test. Therefore, 2-buten-4-olide was found to improve the spatial performance, and this effect is mediated, at least in part, by acidic fibroblast growth factor.

4-Butyrolactone↗

PubMed2001

Fetal alveolar epithelial cells contain [D-Ala(2)]-deltorphin I-like immunoreactivity: delta- and mu-opiate receptors mediate opposite effects in developing lung.

Opiate-like peptides can regulate many cellular functions. We now map [D-Ala(2)]deltorphin I (DADTI)-like immunoreactivity (DADTI-LI) in developing mouse lung and analyze potential functional roles. Most DADTI-LI-positive cells were alveolar cells negative for prosurfactant protein (proSP)-C immunoreactivity. Peak numbers of DADTI-LI-positive cells occurred on embryonic Day 18, decreasing postnatally. To analyze developmental effects of DADTI, e17-18 lung explants were treated with [D-Ala(2)]deltorphin II (DADTII, soluble DADTI analogue, delta-receptor-specific) versus dermorphin (mu-receptor-specific). Type II pneumocyte differentiation, assessed by [(3)H]choline incorporation into saturated phosphatidylcholine and proSP-C immunostaining, was inhibited by DADTII but stimulated by dermorphin. Cell proliferation, measured as [(3)H]-thymidine incorporation and proliferating cell nuclear antigen immunostaining, was stimulated by DADTII and inhibited by dermorphin. All effects were dose-dependent. DADTII-inhibited choline incorporation was reversed by the delta-blocker, naltrindole. Unexpectedly, DADTII-stimulated thymidine incorporation was augmented by naltrindole and reversed by naloxone (mu-blocker). Although dermorphin-stimulated choline incorporation was appropriately blocked by binaltorphimine, dermorphin-inhibited thymidine incorporation was reversed by delta, kappa-, or mu-blockers. The delta- and mu-receptor messenger RNAs occurred pre- and postnatally, whereas kappa-receptor transcripts occurred mainly prenatally. All three receptor proteins were present in epithelial and mesenchymal cells in e18 lung. Thus, DADTI-LI from proSP-C-immunonegative alveolar cells could regulate development via both direct and indirect effects involving multiple opiate receptors.

Animals↗

PubMed2000

Production and immunohistochemical application of antiserum against Tyr-D-Ala-Phe, a N-terminal tripeptide common to dermorphin/deltorphin family.

Tyr-D-Ala-Phe is a N-terminal sequence commonly found in a peptide family including dermorphin and deltorphin. The tripeptide was synthesized and conjugated with poly L-lysine. Nuclear magnetic resonance (NMR) indicated that approximately 38 molecules of the tripeptide were bound to each molecule of poly L-lysine. The conjugate was used to immunize rabbits, and high titer antisera were obtained. An IgG fraction was purified by protein G affinity chromatography. A specific antibody to the tripeptide was then obtained by affinity chromatography using formylcellulofine conjugated with Tyr-D-Ala-Phe. On immunospot assay, the best IgG antibody was capable of detecting 125 ng of Tyr-D-Ala-Phe but failed to react even with 2.0 microg of Tyr-L-Ala-Phe or poly L-lysine. Our immunohistochemical examination selectively localized the secretory glands of frog skin.

Alanine↗

PubMed2000

Production of monoclonal antibody to deltorphin-I and its immunocytochemical application to adult rat brain and cultured rat brain neurons.

A monoclonal anti-deltorphin-I antibody specifically recognizing its NH2-terminal region was produced. In the adult rat brain sections, it recognized immunoreactive nerve fibers mainly in the bed nucleus of stria terminalis, central nucleus of amygdala, lateral hypothalamus, hippocampus, substantia nigra, periaqueductal gray and locus ceruleus. Occasionally, positive somata were localized in the bed nucleus of stria terminalis, central nucleus of amygdala, supraoptic and periventricular nuclei. In primarily cultured neurons from various brain regions of new-born rats, the antibody immunostained strongly neuronal somata and processes. The abundant DADTI-immunoreactive substance in the cultured neurons promises to provide an alternative pathway to search for the counterpart of deltorphins in mammals.

Amygdala↗

PubMed2000

C-type natriuretic peptide-like immunoreactivity in the rat inner ear.

C-type natriuretic peptide (CNP) is a member of the atrial natriuretic peptide family (ANP family). The family also includes ANP and brain natriuretic peptide (BNP). These peptides regulate the homeostasis of body fluid and blood pressure as a neuropeptide in the central nervous system as well as a cardiac hormone in the periphery. We have recently reported the expression of CNP mRNA in the inner ear. To assess the possible physiological role of CNP in the inner ear, we investigated the localization of CNP peptide in the rat inner ear by immunohistochemistry at the light and electron microscopic level. CNP-like immunoreactivity was widely distributed in the secretory and the neuronal portion of the inner ear, i.e. the spiral ligament, the dark cell region of the utriculus, the epithelium of the endolymphatic sac, the spiral ganglion cells and the vestibular ganglion cells. The results suggest that CNP may play a role in the homeostasis of the perilymph and endolymph and may also influence nerve activities in the inner ear.

Animals↗

PubMed2000

A protein encoded by an alternative splice variant of choline acetyltransferase mRNA is localized preferentially in peripheral nerve cells and fibers.

Central cholinergic systems have been visualized by immunohistochemistry using antibodies to choline acetyltransferase (ChAT). Peripheral cholinergic cells and fibers, however, have been hardly detectable with most of these antibodies. This phenomenon suggests that a different form of ChAT may exist in peripheral tissues. Here we report two types of mRNA for ChAT expressed by alternative splicing in rat pterygopalatine ganglion. One is exactly identical with ChAT mRNA reported in the central nervous system (ChAT of a common type; cChAT). The other lacks exons 6, 7, 8 and 9, which was detected only in the pterygopalatine ganglion (ChAT of a peripheral type; pChAT). The peculiarity of pChAT in chemical structure, possessing a splice joint of the exons 5 and 10, led us to produce rabbit antisera against a recombinant peptide of 41 amino acids which spans over the splice joint. On Western blots using a successfully obtained antiserum, an intense band of about 50 kDa, corresponding to the expected molecular weight of pChAT, was detected in the pterygopalatine ganglion but not in the brain. Immunohistochemistry using the antiserum failed to reveal positive staining of known brain cholinergic structures, while it permitted us to observe peripheral, probably cholinergic, nerve cells and fibers including those in the pterygopalatine ganglion and enteric nervous system.

Alternative Splicing↗

PubMed2000

Ontogeny of (D-Ala(2))-deltorphin I-like immunoreactive neurones in foetal rat brain.

Foetal rat brain from embryonic day (ED) 12-22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of (D-Ala(2))-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15-17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation.

Analgesics, Opioid↗

PubMed2000

Immunohistochemical demonstration of choline acetyltransferase of a peripheral type (pChAT) in the enteric nervous system of rats.

Using a recently developed antiserum against a splice variant (pChAT) of choline acetyltransferase, the enzyme which synthesizes acetylcholine, we carried out an immunohistochemical examination in the digestive canal of rats. Positive staining was exclusively localized to neuronal cells and fibers. Positive somata were distributed widely in the intramural ganglia throughout the digestive tract from the esophagus to the rectum. Double staining indicated that, in the rat, virtually all pChAT immunoreactive somata exhibited histochemical activity for acetylcholinesterase but not for NADPH-diaphorase. In the guinea pig, however, there were a few neurons possessing both pChAT and NADPH-diaphorase. We also found a few neuronal somata which were positive for acetylcholinesterase but not for pChAT. The results suggest that pChAT immunohistochemistry is useful for studying the enteric cholinergic system.

Acetylcholinesterase↗

PubMed2000

Enhancement of progenitor cell division in the dentate gyrus triggered by initial limbic seizures in rat models of epilepsy.

PURPOSE: Mitogenic effects of seizures on granule cell progenitors in the dentate gyrus were studied in two rat models of epilepsy. We investigated which stage of epileptogenesis is critical for eliciting progenitor cell division and whether seizure-induced neuronal degeneration is responsible for the enhancement of progenitor cell division. METHODS: Seizures were induced by either kainic acid (KA) administration or electrical kindling. Neurogenesis of dentate granule cells was evaluated using the bromodeoxyuridine (BrdU) labeling method, and neuronal degeneration was assessed by in situ DNA fragmentation analysis. RESULTS: After injection of KA, the number of BrdU-positive granule cells began to increase at day 3 after the treatment, peaked at day 5, and returned to baseline at day 10. By day 13, the values were lower than control. After kindling, the number of BrdU-positive cells began to increase after five consecutive experiences of stage I seizures. The increase occurred from day 1 to day 3 after the last electrical stimulation, but returned to baseline by day 7. After generalized seizures were well established, repeated stimulation did not facilitate division of granule cell progenitors. DNA fragmentation was noted in pyramidal neurons in the CA1, CA3, and hilus regions at 18 h after KA injection, but not in the kindling model. CONCLUSIONS: These observations indicate that a mechanism in epileptogenesis boosts dentate progenitor cell division, but progenitor cells may become unreactive to prolonged generalized seizures. Pyramidal neuronal degeneration is not necessary for triggering the upregulation. It is suggested that newly born granule cells may play a role in the network reorganization that occurs during epileptogenesis.

Animals↗

PubMed1999

Kainic acid-induced inducible cyclooxygenase and c-Jun phosphorylation in the rat hippocampal formation.

Inducible cyclooxygenase (COX-2) was transiently induced in the neurons throughout the entire hippocampus between 6 and 24 h after injection of kainic acid (KA). The induction of COX-2 correlated more closely with the induction of c-Jun than with that of c-Fos. Phosphorylated c-Jun was induced 6-12 h after in the CA3 pyramidal neurons, which undergo apoptosis. Almost all of neurons with phosphorylated c-Jun were colocalized with COX-2. These results suggest that COX-2 and c-Jun phosphorylation may participate in KA-induced neurodegeneration.

Animals↗

PubMed1999

Regulation of inner ear fluid in the rat by vasopressin.

The anti-diuretic hormone vasopressin has been shown to be important in regulating inner ear fluid. The diuretic hormone, CNP, and its receptor, ANP-B receptor, may also function in the regulation of inner ear fluid. To determine whether vasopressin directly affects the fluid level, we infused this hormone to rat and assay of V2-AVP receptor mRNA by semiquantitative RT-PCR demonstrated a significantly lower level of this transcript in vasopressin-infused animals than in saline-infused animals. The levels of CNP and ANP-B receptors mRNA, however, were the same in both groups of rats. Results suggest that high plasma levels of vasopressin may be a principal causal factor of endolymphatic hydrops in Meniere's disease, perhaps by down-regulating the number of vasopressin receptors.

Animals↗

PubMed1999

Hydrogen peroxide-induced apoptosis mediated by p53 protein in glial cells.

It is now generally accepted that massive neuronal death due to oxidative stress is a regular feature of brains in neurodegenerative diseases. However, much less attention has been given to the death of glial cells. In this study, we examined p53-sensitive apoptosis of cells by using human glioblastoma A172 cells and p53-deficient mouse astrocytes. In human A172 cells, hydrogen peroxide (H2O2) caused cell death in a time- and concentration-dependent manner, accompanied by nucleosomal DNA fragmentation and chromatin condensation. After treatment with H2O2, p53 protein was highly expressed and protein levels of Bak, p21WAF1/CIP1 and GADD45 were also enhanced. However, the protein levels of Bcl-2 and Bax did not change. On the other hand, primary cultured astrocytes from p53-deficient mouse brain grew faster than wild-type and heterozygous astrocytes. In addition, p53-deficient astrocytes were more resistant to H2O2-induced apoptosis than wild-type and heterozygous astrocytes. These results suggest that glial proliferation and the repair of damaged DNA may be regulated by p53-induced p21WAF1/CIP1 and GADD45, and that glial apoptosis caused by oxidative stress may be mediated by p53-induced Bak.

Animals↗

PubMed1999

Interferon-gamma plus lipopolysaccharide induction of delayed neuronal apoptosis in rat hippocampus.

Interferon-gamma and lipopolysaccharide (IFN-gamma/LPS) induce expression of inducible nitric oxide synthase (iNOS) protein both in cells in vitro and in the brain in vivo. In cultured cells, excessive production of nitric oxide (NO) induces neuronal cell death. However, it is still unclear whether IFN-gamma and LPS might induce neuronal cell death in vivo. In this study, we examined the neuronal cell death and induction of major histocompatibility complex (MHC) antigens after microinjection of IFN-gamma/LPS into the rat hippocampus. Although microglia appeared morphologically ramified in the normal and vehicle-injected hippocampus, microinjection of IFN-gamma/LPS immediately induced the ameboid type. From days 1-7, iNOS was expressed in ameboid microglia surrounding the site of the microinjection. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells appeared among the granular neurons of the dentate gyrus on day 3 and peaked about 7 days after microinjection. When the NOS inhibitor N(G)-nitro-L-arginine (L-NA) was intraperitoneally administered prior to the microinjection, the number of TUNEL-positive neurons decreased in a L-NA dose-dependent manner. These results suggest that IFN-gamma/LPS induces delayed neuronal apoptosis in the hippocampus in vivo, and it possibly involves excessive NO production by iNOS. Thus, this animal model may be one of neurodegenerative with extensive inflammatory activation in the hippocampus.

Animals↗

PubMed1999

Increased expression of hippocampal cholinergic neurostimulating peptide-related components and their messenger RNAs in the hippocampus of aged senescence-accelerated mice.

Hippocampal cholinergic neurostimulating peptide stimulates cholinergic phenotype development by inducing choline acetyltransferase in the rat medial septal nucleus in vitro. Adult senescence-accelerated-prone mice/8, a substrain of the senescence-accelerated-prone mouse, show a remarkable age-accelerated deterioration in learning and memory. We cloned mouse hippocampal cholinergic neurostimulating peptide precursor protein complementary DNA. The deduced amino acid sequence showed that the neurostimulating peptide itself is the same as that found in the rat. In situ hybridization revealed that the highest expression of the precursor protein messenger RNA was in hippocampal pyramidal neurons. Compared with a strain of senescence-accelerated-resistant mouse (control mouse), adult senescence-accelerated-prone mice/8 showed increased expression of both the precursor messenger RNA and the neurostimulating peptide-related immunodeposits in the hippocampal CA1 field. The deposits were intensely and diffusely precipitated in neuropils throughout the strata oriens and radiatum in senescence-accelerated-prone mice/8, but not in control mice. The neurostimulating peptide content in the hippocampus was higher in senescence-accelerated-prone mice/8 than in control mice, while its precursor protein itself was not different between the two strains. Furthermore, our previous and present data show that the medial septal and hippocampal choline acetyltransferase activity was significantly lower in senescence-accelerated-prone mice/8 than in control mice. The data suggest that, in hippocampal neurons in adult senescence-accelerated-prone mice/8, the production of hippocampal cholinergic neurostimulating peptide precursor protein in neuronal somata, which is associated with an increased expression of its messenger RNA in the CA1 field, occurs as a consequence of low activity in their presynaptic cholinergic neurons. This is followed by accelerated processing to generate bioactive peptide and transport to its functional fields. However, certain mechanisms reduce the release of the peptide and lead to its accumulation in the neuropil. These disturbances of the septohippocampal cholinergic system might be the biochemical mechanism underlying the characteristic deterioration of senescence-accelerated-prone mice/8.

Aging↗