Satoshi Wakisaka

The distribution and origin of substance P-like immunoreactivity in the rat molar pulp and periodontal tissues.

Rat mandibles were fixed in Zamboni fixative and demineralized in a mixture of EDTA and fixative. Substance P-like immunoreactivity was demonstrated by indirect immunofluorescence in molar pulp, periodontal ligament and gingiva. Substance P (SP) containing nerve fibres with varicosities were observed in the pulp horn and root pulp in general located around blood vessels. Some SP-containing fibres penetrated into the predentine and dentine. In the periodontal ligament, SP fibres were localized along the blood vessels in the middle and apical regions. Many SP-containing fibres were associated with the blood vessels in the lamina propria of gingiva. After inferior alveolar nerve section, SP-positive nerve fibres in the pulp and periodontal ligament disappeared completely. In gingiva the number of SP fibres decreased but not all fibres disappeared. Removal of the superior cervical ganglion did not affect the distribution of SP-containing nerve fibres.

The Ruffini Ending as the Primary Mechanoreceptor in the Periodontal Ligament: Its Morphology, Cytochemical Features, Regeneration, and Development

The periodontal ligament receives a rich sensory nerve supply and contains many nociceptors and mechanoreceptors. Although its various kinds of mechanoreceptors have been reported in the past, only recently have studies revealed that the Ruffini endings--categorized as low-threshold, slowly adapting, type II mechanoreceptors--are the primary mechanoreceptors in the periodontal ligament. The periodontal Ruffini endings display dendritic ramifications with expanded terminal buttons and, furthermore, are ultrastructurally characterized by expanded axon terminals filled with many mitochondria and by an association with terminal or lamellar Schwann cells. The axon terminals of the periodontal Ruffini endings have finger-like projections called axonal spines or microspikes, which extend into the surrounding tissue to detect the deformation of collagen fibers. The functional basis of the periodontal Ruffini endings has been analyzed by histochemical techniques. Histochemically, the axon terminals are reactive for cytochrome oxidase activity, and the terminal Schwann cells have both non-specific cholinesterase and acid phosphatase activity. On the other hand, many investigations have suggested that the Ruffini endings have a high potential for neuroplasticity. For example, immunoreactivity for p75-NGFR (low-affinity nerve growth factor receptor) and GAP-43 (growth-associated protein-43), both of which play important roles in nerve regeneration/development processes, have been reported in the periodontal Ruffini endings, even in adult animals (though these proteins are usually repressed or down-regulated in mature neurons). Furthermore, in experimental studies on nerve injury to the inferior alveolar nerve, the degeneration of Ruffini endings takes place immediately after nerve injury, with regeneration beginning from 3 to 5 days later, and the distribution and terminal morphology returning to almost normal at around 14 days. During regeneration, some regenerating Ruffini endings expressed neuropeptide Y, which is rarely observed in normal animals. On the other hand, the periodontal Ruffini endings show stage-specific configurations which are closely related to tooth eruption and the addition of occlusal forces to the tooth during postnatal development, suggesting that mechanical stimuli due to tooth eruption and occlusion are a prerequisite for the differentiation and maturation of the periodontal Ruffini endings. Further investigations are needed to clarify the involvement of growth factors in the molecular mechanisms of the development and regeneration processes of the Ruffini endings.

Cell size analysis of primary neurons innervating the cornea and tooth pulp of the rat

&NA; Primary neuronal cell bodies, whose peripheral axons comprised the cutaneous branch of the mylohyoid nerve or innervated the mandibular molar tooth pulp or the cornea, were labeled with HRP and their cross‐sectional area was analyzed. Most of their cell bodies were smaller than 1000 &mgr;m2 in cross‐sectional area and the histogram of each showed a unimodal pattern. The modes of percentage distribution were 100–200 &mgr;m2 (34.4%), 500–600 &mgr;m2 (17.4%) and 300–400 &mgr;m2 (35.1%) for the mylohyoid nerve, the tooth pulp and the cornea, respectively. A comparison of the 3 histograms indicated that there were at least 3 subpopulations of trigeminal primary neurons i.e., small, medium, and large cells. Electron microscopically, the large primary neurons innervating the tooth pulp had endoplasmic reticulum throughout the cytoplasm. The small primary neurons innervating the cornea showed a clear zonation of organelles and the endoplasmic reticulum was located in the periphery of the cytoplasm. The light microscopically identified small, medium and large cell groups may correspond to C‐, A&dgr;‐ and A&bgr;‐fibers. The tooth pulp and the cornea appear to receive mainly A&bgr;‐fibers and A&dgr;‐fibers, respectively. The cutaneous branch of the mylohyoid nerve appears to contain numerous C‐fibers and progressively smaller proportions of A&dgr;‐ and A&dgr;‐fibers.

Neuropeptides in the dental pulp: Distribution, origins, and correlation*

It is known that many factors participate in the process of inflammation and pain transmission. Recently, various neuropeptides have been demonstrated in the intrapulpal nerve fibers immunobiohistochemically. These neuropeptides may play some role in the process of inflammation and transmission of pain sensation. In this presentation, the distribution, origins, and correlation of neuropeptides in the dental pulp is reviewed.

Peptidergic innervation of the temporomandibular disk in the rat.

The peptidergic innervation of the temporomandibular disk was investigated in the postnatal young rat by using an indirect immunofluorescence method. Calcitonin gene-related peptide-containing nerve fibers were located around the blood vessels and terminated as free nerve endings in the disk. These nerve fibers may be of a sensory nature.

Cartducin, a paralog of Acrp30/adiponectin, is induced during chondrogenic differentiation and promotes proliferation of chondrogenic precursors and chondrocytes

We previously reported that CORS26 gene, isolated from C3H10T1/2 cells treated with transforming growth factor‐β1, was predominantly expressed in cartilage. Because the gene product is a kind of secretory protein produced by cartilage tissue, we named it “cartducin”. Cartducin shares a similar modular organization to adipocyte‐derived hormone, adiponectin. In this study, we investigated cartducin function during chondrogenesis and cartilage development. In situ hybridization analysis showed that cartducin transcripts were restricted to the proliferating chondrocytes in the growth plate cartilage. Whole‐mount in situ hybridization revealed that the first significant induction of cartducin expression occurred in the sclerotome, which contains a chondrogenic cell lineage between days 9.5 and 10.5 postcoitus (p.c.) during mouse embryogenesis. Chondrogenic differentiation by combined treatment with bone morphogenetic protein‐2 and insulin induced cartducin expression along with type II and IX collagen expression in chondrogenic progenitor N1511 cells. To elucidate the direct action of cartducin on the cells, recombinant cartducin protein was expressed in and purified from Escherichia coli. The recombinant cartducin potentially forms homo‐oligomers and promoted the proliferation of chondrogenic progenitor N1511 cells, and chondrocytic HCS‐2/8 cells in a dose‐dependent manner. On the other hand, cartducin did not affect the production of sulfated glycosaminoglycan (sGAG) in these cells. These findings indicate that cartducin is a novel growth factor and plays important roles in regulating both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes. J. Cell. Physiol. 206: 537–544, 2006.

Substance P-like immunoreactivity in the pulp-dentine zone of human molar teeth demonstrated by indirect immunofluorescence

Freshly-extracted human third molars were fixed in Zamboni fixative, demineralized with the mixture of EDTA and the fixative; substance P-like immunoreactivity (SPLI) was revealed by the indirect immunofluorescence technique of Coons. Substance P (SP) was observed in the pulp-dentine zone and the dental pulp. Some of SP-containing fibres ended at the odontoblast layer and did not reach the predentine; others terminated at the predentine surface or penetrated into the predentine. In the predentine, some of SP fibres accompanied odontoblast processes and ended near the mineralized dentine; others changed course transversely at various levels.

Cell size-specific appearance of neuropeptide Y in the trigeminal ganglion following peripheral axotomy of different branches of the mandibular nerve of the rat

The effect of peripheral axotomy of the mental nerve (MN) and the cutaneous branch of the mylohyoid nerve (MhN) on the appearance of neuropeptide Y-like immunoreactivity (NPY-IR) in cells in the trigeminal ganglion of the rat was examined with combined retrograde-tracing and immunohistochemistry. Retrograde-tracing with True Blue (TB) revealed that the cell-size spectrum of the trigeminal cells sending peripheral processes to the MN (TB MN cells) ranged from 75.9 to 1560.5 microns2 (or from 9.8 to 44.6 microns in diameter); approximately 53% of TB MN cells were 300-600 microns2. TB MhN cells ranged from 47.7 to 1261.5 microns2 (or from 7.8 to 40.1 microns in diameter); 56% of TB MhN cells were < 300 microns2. In the normal trigeminal ganglion, there were no NPY-IR cells. 14 days after MN transection, approximately 35% of TB MN cells displayed NPY-IR. The distribution of the cross-sectional areas of NPY-IR cells after MN transection was very similar to that of TB MN cells. Transection of MhN also induced the appearance of NPY-IR in the trigeminal ganglion but to a lesser extent (approximately 17% of TB MhN cells). The distribution of the cross-sectional areas of NPY-IR cells after MhN transection was similar to that of NPY-IR cells after MN transection. These results indicate that injury-evoked NPY-IR is specific for the medium- and large-sized ganglion cells.

NADPH-diaphorase in the developing rat: Lower brainstem and cervical spinal cord, with special reference to the trigemino-solitary complex

A previous study indicated that in adult rat, a distinctive neuronal group in the dorsomedial division of the subnucleus oralis of the spinal trigeminal nucleus (SpVo) and the rostrolateral part of the nucleus of the solitary tract (Sn) is stained for nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d), and suggested that the labeled structures are involved with sensorimotor reflexive functions. This study aimed to characterize the developmental expression of NADPH‐d in SpVo and Sn, including other areas of the lower brainstem and cervical spinal cord, by means of the enzyme histochemical staining technique, from the prenatal through the postnatal period. On embryonic day 12 (E12), no neurons in the brain were stained for NADPH‐d, whereas blood vessels were stained. Labeling in the vessels was consistently present throughout pre‐ and postnatal periods but decreased with development. On E15, labeled neurons appeared in the dorsomedial part of SpVo and the rostrolateral part of Sn, but not in the other nuclei. The labeled neurons in both nuclei increased in numbers drastically to E17. Postnatally, they tended to increase gradually in Sn, but to decrease slightly in SpVo. The cell size of labeled neurons reached a plateau at E17 in SpVo, but at postnatal day 4 (P4) in Sn. In other nuclei on E17, labeling appeared in the lateral paragigantocellular reticular, intermediate reticular, medullary reticular, pedunculopontine tegmental, and spinal vestibular nuclei, and laminae V, VI, and X of the cervical spinal cord. On E20 and P0, labeling appeared in the dorsal column, laterodorsal tegmental, raphe obscurus, parvocellular reticular, ventral gigantocellular reticular, and parahypoglossal nuclei, and laminae IX of the cervical spinal cord. On P4, labeling appeared in the parabrachial and median raphe nuclei, medial and caudolateral Sn, the magnocellular zone of subnucleus caudalis of the spinal trigeminal nucleus (SpVc), and laminae III/IV of the cervical spinal cord. On P10, labeling appeared in the paratrigeminal and dorsal raphe nuclei, the superficial zone of SpVc, and laminae I/II of the cervical spinal cord. No newly labeled neurons appeared in any nuclei after P14.

CTRP3/cartducin promotes proliferation and migration of endothelial cells

CTRP3/cartducin, a novel secretory protein, is a member of the C1q and tumor necrosis factor (TNF)-related protein (CTRP) superfamily. CTRP3/cartducin gene is transiently up-regulated in a balloon-injured rat carotid artery tissue. In this study, we report a new function of CTRP3/cartducin as a regulator of angiogenic processes. CTRP3/cartducin promoted proliferation and migration of mouse endothelial MSS31 cells in a dose-dependent manner. Further, stimulation of MSS31 by CTRP3/cartducin led to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). MAPK/ERK kinase 1/2 (MEK1/2) inhibitor, U0126, and p38 MAPK inhibitor, SB203580, blocked the CTRP3/cartducin-induced cell proliferation, and migration was blocked by U0126, but not the SB203580. Taken together, these results suggest that CTRP3/cartducin may be involved as a novel angiogenic factor in the formation of neointima following angioplasty.