J. Kato

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.

Distribution and origin of nerve fibers in the rat temporomandibular joint capsule.

Abstract The distribution and origin of nerve fibers containing neuropeptides and NOS projecting to the temporomandibular joint capsule (TMJ) of the rat were studied by retrograde tracing in combination with immunocytochemistry. Numerous nerve fibers were seen in the TMJ as revealed by the neuronal marker protein gene product 9.5. Nerve fibers containing neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), pituitary adenylate cyclase activating peptide (PACAP), substance P (SP), calcitonin gene-related peptide (CGRP), and nitric oxide synthase (NOS) were seen in the synovial membrane, the joint capsule and entering the articular disc. Injection of the retrograde tracer True Blue (TB) into the TMJ resulted in the appearance of numerous labeled nerve cell bodies in the trigeminal and superior cervical ganglia, and moderate numbers in the nodose, the otic, the sphenopalatine, the stellate and the dorsal root ganglia at levels C2–C5. Most of the TB-labeled cell bodies in the superior cervical and stellate ganglia contained NPY. In the trigeminal ganglion, numerous TB labeled cell bodies contained CGRP and a minor population stored SP, a few cell bodies were seen to store NOS or PACAP. In the sphenopalatine and otic ganglia, TB labeled cell bodies contained NOS or VIP. In the nodose ganglion, labeled cell bodies contained CGRP; other labeled cell bodies harbored NOS. In the cervical dorsal root ganglia, the majority of the labeled cell bodies stored CGRP and smaller populations stored SP and PACAP. Thus, the innervation of the TMJ is complex and many different ganglia are involved.

Protein gene-product 9.5 in developing mouse circumvallate papilla: comparison with neuron-specific enolase and calcitonin gene-related peptide

The present study was made to investigate the ontogeny of protein gene-product 9.5 (PGP 9.5)-like immunoreactivity (-LI) in the developing mouse circumvallate papilla (CVP), and its distribution was compared to that of neuron-specific enolase (NSE) and calcitonin gene-related peptide (CGRP). In adult CVP, PGP 9.5-LI was observed in the subgemmal nerve plexus; some thin PGP 9.5-like immunoreactive (-IR) nerve fibers penetrated taste buds and apical epithelium. PGP 9.5-LI was also observed in the spindle-shaped cells in taste buds, and a small number of round- or oval-shaped ganglionic cells in the lamina propria. The distribution of NSE-LI was comparable to that of PGP 9.5-LI. CGRP-LI was observed in the nerve fibers only; distribution of CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers, although the number of CGRP-IR nerve fibers was smaller than that of PGP 9.5-IR nerve fibers. At least six developmental stages were defined with regard to the developmental changes in the distribution of PGP 9.5-LI from embryonic day (E) 12 to adulthood: Stage I (E12–13) — a dense nerve plexus of PGP 9.5-IR nerve fibers was detected in the lamina propria beneath the core of newly-formed papilla. Stage II (E14–16) — thin PGP 9.5-IR nerve fibers penetrated the apical epithelium, and a few round-shaped cells in the apical epithelium also displayed PGP 9.5-LI. Stage III (E17–18) — thin PGP 9.5-IR nerve fibers penetrated the inner lateral epithelium of the trench. Stage IV [Postnatal day (P) 0–3] many PGP 9.5-IR nerve fibers penetrated the outer lateral epithelium of the trench; later in this stage, taste buds appeared. Stage V (P5–10) — a small number of PGP 9.5 IR cells in the taste buds appeared, and their number increased gradually. Stage VI (PI4-adult) — the number of PGP 9.5-IR taste cells increased and reached the adult level, while the number of PGP 9.5-IR nerve fibers decreased. The development of NSE-LI was similar to that of PGP 9.5-LI. CGRP-IR nerve fibers were detected at E12 in the lamina propria, and the development of the intraepithelial CGRP-IR nerve fibers was similar to that of PGP 9.5-IR nerve fibers. The present results indicate that invasion by nerve fibers of the epithelium of lingual papillae occurs in a complex manner, and that these nerve fibers may participate in the formation of the taste buds.

IMMUNOHISTOCHEMICAL CHANGES IN THE DISTRIBUTION OF NERVE FIBERS IN THE PERIODONTAL LIGAMENT DURING AN EXPERIMENTAL TOOTH MOVEMENT OF THE RAT MOLAR

We examined the distribution of nerve fibers containing neurofilament protein (NFP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY) in the periodontal ligament (PDL) of the rat first molar. In addition, we observed changes in the distribution of these nerve fibers in the PDL during experimental tooth movement induced by insertion of a separating elastic. In normal animals, both NFP- and CGRP-containing nerve fibers were observed in the apical part of the PDL, smaller numbers being found in the cervical part in normal rats. They terminated as free nerve endings in the apical part; no specialized endings were observed. VIP- and NPY-containing nerve fibers were associated with blood vessels near the alveolar bone in the apical part of the PDL of normal animals. Three days after tooth movement, the number of NFP- and CGRP-containing nerve fibers increased in both stretched and compressed parts of the PDL, and returned to normal levels after 14 days. Immediately after tooth movement, VIP-containing nerve fibers disappeared in the stretched part, and returned to the normal level after 14 days. VIP-containing nerve fibers were always observed during tooth movement in the compressed part of the PDL. NPY-containing nerve fibers disappeared immediately after tooth movement in the stretched part and returned to the normal level after 3 days. NPY-containing nerve fibers were not observed 1 day after insertion of the elastic in the compressed part and were the same as in the normal controls after 14 days. These results suggest that NFP-, CGRP-, VIP- and NPY-containing nerve fibers in the PDL play important roles in the modulation of pain, tissue remodeling and blood flow regulation during tooth movement.

Induction of Fos protein in the rat trigeminal nucleus complex during an experimental tooth movement

The induction and temporal changes of Fos protein in the rat spinal trigeminal nucleus complex during experimental tooth movement were studied immunohistochemically. Separating elastics were unilaterally inserted between the upper molars. The animals were perfused at 0, 1, 2, 4, 8, 12 and 24 h thereafter, and the brains containing the mesencephalic trigeminal nucleus and the spinal trigeminal nucleus complex were then removed. Cells showing Fos immunoreactivity were observed in the superficial layers of the subnucleus caudalis on the ipsilateral side except at 0 and 24 h. The numbers of the immunoreactive cells peaked 2 and 4 h after the insertion. On the contralateral side, only a few immunoreactive cells were observed in the subnucleus caudalis of the 1-, 2- and 4-h groups. The subnucleus interporalis and the subnucleus oralis of the spinal trigeminal nucleus complex, the principal trigeminal nucleus and the mesencephalic trigeminal nucleus of the experimental animals and all the trigeminal nuclei of the control animals contained no immunopositive cells. Thus experimental tooth movement induced a Fos protein in the superficial layers of the subnucleus caudalis of the spinal trigeminal nucleus complex. The subnucleus caudalis may modulate pain induced by tooth movement.

Parathyroid hormone-related peptide is involved in protection against invasion of tooth germs by bone via promoting the differentiation of osteoclasts during tooth development

In order to elucidate the role of parathyroid hormone-related peptide (PTHrP) in tooth development, we treated tooth germ explants of mouse molars with antisense phosphorothioate-oligodeoxynucleotide (ODN) against PTHrP. Antisense ODN-treatment of the explants resulted in the invasion of the tooth germs by bone. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells around the tooth germs in antisense ODN-treated explants was much lower than that of the control explants. Electron microscopic examination suggested that the antisense ODN-treatment inhibited differentiation of osteoclasts. Treatment of the explants with bisphosphonate or vitamin K2, inhibitors of the differentiation of osteoclasts, induced the invasion by bone into the tooth germs as observed in the antisense ODN-treated explants. The results obtained suggest that PTHrP is involved in the mechanism protecting tooth germs from bone invasion by promoting the differentiation of osteoclasts around them.

Neuropeptide Y-immunoreactive primary afferents in the dental pulp and periodontal ligament following nerve injury to the inferior alveolar nerve in the rat.

The distribution of neuropeptide Y (NPY)-immunoreactive (IR) primary afferents in the dental pulp and periodontal ligament of the rat mandible were examined following combined chronic constriction injury (CCI) of the inferior alveolar nerve (IAN) and sympathectomy of the superior cervical ganglion (SCG). NPY-IR nerve fibers were observed around the blood vessels in the trigeminal ganglion, dental pulp and periodontal ligament in normal animals. Following combined CCI of the IAN and sympathectomy of SCG (SCGx), perivascular NPY-IR nerve fibers originating from SCG disappeared completely, but many NPY-IR nerve fibers coming from the trigeminal ganglion appeared in the dental pulp and periodontal ligament. In the molar dental pulp, thick NPY-IR nerve fibers were observed within the nerve bundle, and some thin NPY-IR nerve fibers ran towards the odontoblast layer; very few NPY-IR nerve fibers were observed in the incisor pulp. In the periodontal ligament of molar, thick NPY-IR nerve fibers appeared at the alveolar part following combined CCI of IAN and SCGx. In the lingual portion of the periodontal ligament of the incisor, many thick NPY-IR nerve fibers were observed. These occasionally showed a tree-like appearance, resembling immature Ruffini endings; slowly adapting mechanoreceptors. The present results indicate that periodontal mechanoreceptors are among the main targets of injury-evoked NPY following IAN injury.

Appearance of neuropeptide Y-like immunoreactive cells in the rat trigeminal ganglion following dental injuries

The effects of these injuries on the presence and distribution of neuropeptide Y (NPY)-immunoreactive (-IR) neurones were examined immunohistochemically. In the normal trigeminal ganglion: some perivascular nerves displayed NPY-IR but there were no NPY-IR ganglionic cells. Fourteen days after extraction or pulp exposure of the upper first molar, NPY-IR cells appeared in the maxillary region of the trigeminal ganglion. About 90% of the injury-evoked NPY-IR cells had medium to large diameters (more than 300 microns2 in cross-sectional area). Shallow cavity preparation, however, did not induce the appearance of NPY-IR cells in the trigeminal ganglion. These results indicate the dental injuries alter the primary sensory neurones in the trigeminal ganglion.

Tooth-type specific expression of dHAND/Hand2: possible involvement in murine lower incisor morphogenesis.

Abstract. dHAND/Hand2 is a basic helix-loop-helix transcription factor required for the development of the heart, pharyngeal arches, and vasculature and is expressed during embryogenesis. However, there are no reports on the involvement of the dHAND gene in tooth development. In the present study, the expression of dHAND was examined in developing tooth germs of mice. The dHAND gene was expressed in the mesenchyme of the presumptive incisor region of the lower jaw at an early stage and in the mesenchyme of the lower incisor tooth germ at a later stage. However, the dHAND gene was not expressed in the upper incisor region or the upper and lower molar regions during jaw development. Treatment of tooth germ explants of lower incisors with antisense oligodeoxinucleotide (ODN) against dHAND prevented the differentiation of tooth germ cells, including ameloblasts and odontoblasts, the formation of dentin and enamel, and the proliferation of tooth germ cells and increased the apoptosis of tooth germ cells, suggesting that dHAND is essential for these cells during development. On the other hand, the treatment of tooth germ explants of upper incisor and upper or lower molars did not induce severe effects on their development. Treatment of the explants with basic fibroblast growth factor in association with antisense ODN partially rescued them from the effects of antisense ODN. The present results suggest that the dHAND gene plays important roles in type-specific development of lower incisors, and that basic fibroblast growth factor is involved downstream of the dHAND pathway in tooth germ cells.

Expression of calcitonin gene-related peptide-1 receptor mRNA in human tooth pulp and trigeminal ganglion

Numerous nerve fibres containing calcitonin gene-related peptide (CGRP) were found by immunocytochemistry in human molar pulp. These nerves were often seen around small blood vessels and as free endings without vascular contact. In the trigeminal ganglion a large number of CGRP-immunoreactive nerve-cell bodies, mostly of small to medium size, was encountered. Reverse transcriptase-polymerase chain reaction, using specific sense and antisense primers, detected mRNA expression of the human CGRP1 receptor in the pulp tissue and the trigeminal ganglion. Thus, both CGRP-containing nerve fibres and CGRP1 receptor mRNA are present in human tooth pulp, where they may be involved in the regulation of vascular tone and other local reactions to injury.