Hideko Tsuzuki

Immunohistochemical analysis of pulpal innervation in developing rat molars

The beginning of pulpal innervation was examined in these developing teeth. Mandibular first molars removed from newborn to 7-day-old rats were cryosectioned and nerve fibres were localized by the peroxidase-antiperoxidase technique using a neuropeptide-specific (200 kDa) antibody. Some pulps from 5- and 7-day-old rats were also examined by conventional electron microscopy. In newborn to 4-day-old rats, molars in the initial stages of the dentine deposition were innervated in the follicles but not in the pulps. In molars from 5-day-old rats, nerve fibres were found in the pulps of 11 out of 16 samples. The fibres were mainly located along the blood vessels in the basal part of the pulp, with some arborizations. In rats of 6 and 7 days old, nerve fibres were found in all the pulps examined. These had gradually extended deep into the cuspal area and were increasingly arborized with increasing age. Nerve fibres were also found along the basal laminae of blood vessels in some dental pulps from 5-day-old rats when examined by electron microscopy. At 7 days, nerve fibres were composed of bundles of axons, some of which were covered with Schwann cell processes and basal laminae. These observations indicate that the innervation of rat molar dental pulps begins after the start of the deposition of enamel, in animals of around 5 days of age, which is at the same stage as in mouse molars, as others have shown by a silver-staining method.

Enhanced Regeneration of Critical Bone Defects Using a Biodegradable Gelatin Sponge and β-Tricalcium Phosphate with Bone Morphogenetic Protein-2

We examine the osteogenicity of a sponge biomaterial consisting of a biodegradable mixture of gelatin and β-tricalcium phosphate (βTCP) that bound bone morphogenetic protein 2 (BMP-2) in critical-sized bone defects in rats. Gelatin-βTCP sponges containing either phosphate buffered saline or incorporating BMP-2 are implanted into 5 mm diameter bone defects created in rat mandibles. We assess the defects biweekly for 8 weeks following implantation. There is significantly higher osteoinductive activity and significantly more Gla-osteocalcin content at bone-defect healing sites treated with gelatin-βTCP sponges incorporating BMP-2 than there is in those treated with sponges that did not contain BMP-2. Histologically, new bone that contains bone marrow and that is connected to the original bone almost entirely replaces the regenerated bone. These results show that biodegradable gelatin-βTCP incorporating BMP-2 is osteogenic enough to promote healing in large bone defects.

Effects of extrinsic autonomic inputs on expression of c-Fos immunoreactivity in myenteric neurons of the guinea pig distal colon.

c-Fos protein is a nuclear protein coded by c-fos proto-oncogene subsequent to synaptic activation of the neurons. We used immunohistochemical methods to visualize the expression of c-Fos protein in myenteric neurons of the guinea pig distal colon and examined the effects of the extrinsic autonomic inputs on the enteric circuits. No c-Fos immunoreactivity was observed in the colonic segments fixed immediately after removal from the animal body. A number of c-Fos-immunoreactive nuclei of myenteric neurons, however, appeared in all preparations that were incubated in Krebs solution in vitro (n=10). Application of tetrodotoxin (0.2 microM) abolished the expression of c-Fos-immunoreactivity (n=6), but hexamethonium (100 microM) failed to decrease the number of c-Fos-positive neurons despite a complete suppression of spontaneous peristaltic movements (n=5). Neither the electrical stimulation (n=8) nor the severing of the pelvic nerves (n=5) changed the number of c-Fos-positive neurons. Application of clonidine, an alpha(2)-agonist, (0.1 microM) abolished the expression of c-Fos protein in all preparations (n=5), while denervation of the sympathetic fibers in the lumbar colonic and hypogastric nerves in vivo increased the number of c-Fos-positive neurons (n=5). The results indicate that the enteric circuit in the distal part of the gastrointestinal tract is under tonic inhibition by the sympathetic nervous system from the lumbar spinal cord. c-Fos immunoreactivity expressed in the colonic preparations in vivo might be the results of enhanced activation of non-nicotinic receptors after removal of the sympathetic inhibition.

A sperm factor as the counterpart to the sperm-binding factor of the homologous eggs.

Abstract A substance was isolated from sperm of the sea urchin, Hemicentrotus pulcherrimus . When unfertilized eggs of homologous species were pre-treated with this substance, they rapidly lost the fertilizability due to loss of the sperm-binding capacity. Such an effect was not exerted upon eggs either in CaMg free sea water or of heterologous species. This substance caused neither iso-agglutination of eggs nor precipitation of jelly coat. When it was pre-incubated with the sperm-binding factor purified from eggs of homologous species, it lost the fertilization-inhibiting effect on eggs. It seems very likely that a complementary relationship exists between the present substance and the sperm-binding factor.

Cholinergic traits in rat mandibular processes observed by electron microscopy

SummaryCholinergic traits in rat mandibular processes were examined histochemically, under the electron microscope, scope, at early developmental stages (Stages 20 to 23, by Christies nomenclature). The histochemical reaction for detection of enzymes was performed by the thiocholine method. Nonspecific cholinesterase (EC 3.1.1.8) activity was found in ectomesenchymal cells, vascular endothelial cells, and in some epidermal cells at stages 20 and 21. The enzymatic activity was localized in the perinuclear and endoplasmic reticular cisternae. At stage 22, the number of cells with enzymatic activity decreased gradually, except in the case of the capillary endothelial cells. At stage 23, when the trigeminal nerve fiber was obvious in the mandibular processes, nonspecific cholinesterase activity was restricted to some of the endothelial cells and trigeminal ganglionic cells. In contrast, acetylcholinesterase activity was found on the membrane of trigeminal nerve fiber. Thus, the transient, nonspecific, cholinesterase activity, found in rat mandibular processes, may serve some functions in transmission, lipid metabolism or destruction of toxic cholinesters during the period that precedes organogenesis.

S100-immunoreactive cells contacting with rat mesencephalic trigeminal neurons

reduced by administration of the intracellular Ca2+ store depletor, thapsigargin or IP3 receptor antagonist, 2-APB. Therefore, these Ca2+ rhythms were not induced by action potential, and mainly due to Ca2+ release from an intracellular Ca2+ store (i.e. endoplasmic reticulum) via IP3 receptor.Activation of some kind of metabotropic receptors induces the Ca2+ release from an intracellular Ca2+ store. It was reported that metabotropic glutamate receptor type 5 (mGluR5) was one of therapeutic targets for Parkinson’s disease. Thus, we investigated effects of mGluR5 on the spontaneous Ca2+ rhythms. Antagonist of mGluR5, MPEP, suppressed the spontaneous Ca2+ rhythms. Thus, mGluR5PLC-IP3 signal cascade might be concerned with the Ca2+ rhythms. This result suggested that mGluR5 might contribute to the information processing in striatum by means of regulating intracellular Ca2+ concentration. Research fund: JST, CREST.

Glial cells with S100-like immunoreactivity in the rat trigeminal motor nucleus

Cilia are evolutionary conserved organelles that are classified into either conventional (motile) or primary (nonmotile). Compared with the conventional cilia whose functions are well known, the physiological function and significance of primary cilia are largely unknown, except for its pivotal role in early vertebrate development. Recent studies have shown that primary cilia brought together receptors and signal transduction components, such as Wnt and Hedgehog (Hh) signaling pathway. Although previous studies have shown that molecules for Hh signaling cascade are active in various cells during development, their physiological functions in the nervous system, especially in glial cells, are still enigmatic. In this study, we identified primary cilia in several types of glial cells. Moreover, Hh signaling molecules were accumulated on the primary cilia. Our studies indicate that Hh signaling via primary cilia playa an important role in proliferation and survival of glial cells under stressed condition of the cell.

Serotonin-immunoreactive Epithelial Cells in the Main Excretory Ducts of Rat Submandibular Glands

Although several morphological studies of main excretory duct of the submandibular gland have been performed, few reports present immunohistochemical data. Some epithelial cells or basal cells contain dense granules of an array shape by electron microscopic observation. However, the details of granulated cells have not been clarified immunohistochemically. In this study, both protein gene product 9.5 (PGP 9.5) and chromogranin or serotonin immunoreactive cells in the main excretory duct (MED) of the rat submandibular gland were observed. Some nerve endings were also observed among the epithelium of the MED. Therefore, the modifying mechanism of primary saliva in the MED may be regulated by both serotonergic nerve and endocrine cells with serotonin.