Rat Molar Tooth Research Articles

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth.

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.

Localization of nitric oxide synthase in the periodontal tissues of orthodontically moved and stationary teeth

There is ample evidence that the inflammatory response and vascular supply of the periodontal tissues is crucial to mediating the effects of applied force. The connection between mechanical forces and vascular activity, however, is not well defined. Nitric oxide (NO), a molecule only recently recognized as a chemical messenger within biologic systems, has been shown to be central to these processes in many parts of the body, but not during tooth movement. Therefore, the aim of this investigation was to identify the sites of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) activity in the periodontal tissues of moved and stationary teeth immunohistochemically. Our data show that the periodontal tissues of stationary rat molar teeth exhibit the presence of less eNOS and iNOS. There is an increased amount of eNOS produced after rat molar tooth movement in blood vessel walls. Similarly, increased amounts of iNOS produced after rat molar tooth movement in the periodontal ligament and connective tissue between roots of moved teeth as well as around blood vessels could be seen. The presence of eNOS and iNOS was consistently seen in the odontoblast layer of pulp chambers of moved and stationary teeth. Our results indicate a possible role of NO in periodontal remodeling during tooth movement.

Apoptosis in the Connective Tissues of the Periodontal Ligament and Gingivae of Rat Incisor and Molar Teeth at Various Stages of Development

Apoptosis in the periodontal connective tissues was studied using the TUNEL technique, supported by electron microscopy. For 16 rats (aged 3, 8, or 104 weeks), nuclear fragmentation was assessed using the TUNEL technique (for 4 of the animals aged 8 weeks, incisor eruption was experimentally increased by trimming of teeth to the gingival margin--unimpeded eruption). A further 8 rats (aged 8 and 104 weeks) were employed for electron microscopy. For the incisor, prior to aging, and regardless of eruptive behavior (i.e., for both impeded and unimpeded incisors), there was little evidence of apoptosis in the periodontal ligament or gingival connective tissues. For the molar, apoptosis was also not usually detected when the teeth were erupting or in the mature, erupted state. In the aged animals, however, there was a marked increase in apoptosis (as assessed by the TUNEL technique) within the periodontal ligament and gingivae of both the molars and incisors (where eruption rates also increased). Nevertheless, electron microscopy indicated that significant numbers of apoptotic cells were only in the incisor periodontium. These findings are not consistent with the view that the periodontal fibroblasts provide a component of the force(s) responsible for eruption.

The spatial and temporal expression of calretinin in developing rat molars ( Rattus norvegicus)

Calretinin is a 29-kDa calcium-binding protein abundantly expressed in central and peripheral neural tissues. The aim here was to determine its expression during various stages of odontogenesis. Five categories of embryonic (E) and postnatal (P) rats at various ages (E17, E18, E20, P0, and P7), both male and female, were used to represent the various stages of molar tooth development. The heads of the experimental animals were harvested at the appropriate time and each was cut mid-sagittally and coronally to locate the tooth germs. Selected sections were stained immunohistochemically with polyclonal rabbit anticalretinin at a concentration of 1:25 after microwave irradiation. The results showed that calretinin is distributed widely in epithelium-derived tissues during odontogenesis in rat molar tooth germs. It was expressed focally in the dental lamina, outer enamel epithelium, stellate reticulum and stratum intermedium at different stages. In contrast, it was expressed diffusely and intensely in the inner enamel epithelium and presecretory ameloblasts, although it was discontinuous over the cusp tips. In the secretory ameloblasts, the staining was less intense, being restricted to the cytoplasm, including Tomes' processes. This distribution suggests that calretinin may play a part in enamel formation.

Temporal changes in the distribution and number of macrophage-lineage cells in the periodontal membrane of the rat molar in response to experimental tooth movement

In order to evaluate the possible role of macrophages in the remodelling of periodontal tissue in response to tooth movement, temporal changes in the number and distribution of macrophage-lineage cells in the periodontal membrane of the rat molar tooth after experimental tooth movement were examined immunohistochemically using four anti-rat monoclonal antibodies: ED1 (anti-monocyte/macrophage-lineage cells and dendritic cells), ED2 (anti-resident macrophages), KI-M2R (anti-tissue macrophages), and OX6 (anti-classII molecules). The right maxillary first molar tooth of Wistar rats was moved mesially by a closed-coil spring for 1, 3, 5, or 7 days. Sham-treated rats wearing an inactivated appliance for each experimental period and entirely untreated rats were used as controls. Alternate horizontal serial cryostat sections were cut and incubated with antibodies to ED1, ED2, KI-M2R, and OX6. In addition, cells immunopositive for each monoclonal antibody in the periodontal membrane during tooth movement were analysed on the tension and pressure sides. In the control rats, large numbers of cells positively stained with each monoclonal antibody were distributed throughout the periodontal membrane surrounding the distobuccal root. At 1 day after experimental tooth movement, the number of immunopositive cells obtained with all four monoclonal antibodies decreased as compared with those of the control on the mesial/pressure side. During the later experimental time periods, ED1- and OX6-positive cells in the periodontal membrane of this side were significantly increased in number compared with controls, whereas the density and distribution pattern of cells positive with ED2 or KI-M2R remained unchanged. On the mesial/pressure side, which underwent hyalinization, a marked accumulation of OX6- and ED1-reactive cells, but not of ED2- or KI-M2R-reactive cells, was frequently observed in the area of the hyalinized tissue at 5–7 days after the start of tooth movement. On the distal/tension side, no particular change in the distribution of immunopositive cells obtained with any antibody was detected throughout the experimental periods, with the exception that there was a significant increase in the number of ED1-positive cells and in of OX6-positive cells at 1 and 7 days, respectively, after the start of tooth movement. These results suggest that after the start of tooth movement OX6- and ED1-positive cells, which are mostly exudative macrophages, but not ED2- and KI-M2R-positive cells, i.e., resident macrophages, may be actively engaged in bone resorption and the remodelling of tissues on the pressure side of the periodontal membrane.

Arrest of Rat Molar Tooth Development by Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin

The interference with tooth development by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was studied in dioxin-resistant Han/Wistar rats. Lactating dams were given a single dose of 50 or 1000 μg TCDD/kg body wt 1 day after delivery and the pup heads were analyzed radiographically or histologically at postnatal days 9 and/or 22. Of 19 animals studied histologically, 10 lacked one or more third molars, which were at the bud stage at the start of the experiment. A higher proportion of pups exposed to the higher dose (9/13) lacked third molars than those exposed to the lower dose (1/6) (27/52 and 2/24 teeth missing, respectively). Missing upper third molars (19/38) were more frequent than were lower (10/38). The development of the third molars present was retarded. The root tips of the more advanced first and second molars were prematurely closed and root formation was arrested, but eruption was not affected. Dentinogenesis of the continuously erupting lower incisor teeth was preeruptively arrested because of pulpal cell death. All the teeth of the control rat pups developed normally. In contrast to the control pups, none of the 11 experimental pups examined radiographically (6 exposed to the higher dose and 5 to the lower) showed mineralization of their third molar cusps. The results show that the effects of TCDD on rat tooth development depend on not only the dose but also the tooth type and developmental stage. Inasmuch as early tooth development is under the control of inductive interactions between the epithelium and the mesenchyme, the interference by TCDD with tooth morphogenesis with the consequent arrest of development is likely to involve epithelial–mesenchymal signaling.

Morphological and immunohistochemical changes of epithelial tissues of eruprion passage of the rat molar teeth during eruption period.

Morphological and immunohistochemical changes of epithelial tissues of eruprion passage of the rat molar teeth during eruption period.

Highly Regulated Expression of Subtilisin-like Proprotein Convertase PACE4 (SPC4) during Dentinogenesis

Expressions of mRNAs for four subtilisin-like proprotein convertases (SPCs: furin, PACE4, PC6, and PC8) and bone morphogenetic protein 4 (BMP4) in the rat molar tooth during development were analyzed by Northern blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization to explore the possible involvement of SPCs in the processing of proBMPs. We found a temporospacial expression of PACE4, but not one of the other SPCs, in this tissue; i.e., RT-PCR analysis revealed that the level of PACE4 mRNA, but not that of the other SPC mRNAs became high around the second postnatal day. This increase was in good accordance with the increase in BMP4 mRNA, indicating an apparent association of these molecules with the differentiation and establishment of functional ameloblasts and odontoblasts. During dentinogenesis, PACE4 mRNA was localized in the ameloblasts and odontoblasts. These observations suggest that PACE4 plays a crucial role in dentinogenesis, especially via the activation of BMPs.

Calbindin D28k-like immunoreactivity during the formation of the enamel-free area in the rat molar teeth.

Previous studies have demonstrated the presence of calbindin D28k in the ameloblasts derived from the inner enamel epithelium. The occlusal surfaces of the rodent molars partly lack the enamel covering, which is referred to as enamel-free area (EFA). In the present study, we compared the immunohistochemical localization of calbindin D28k-like immunoreactivity (CB-LI) in the cells at the EFA (EFA cells) and ameloblasts of the rat molar teeth at the light microscopic level. CB-LI was strong in the ameloblasts of the presecretory through the protective stages, while it was faint at the late secretory to transitional stages. However, some mature ameloblasts lacked the immunoreactivity. On the other hand, the majority of EFA cells showed distinct polarization and elongation that were absent in few cells at the early stage of EFA formation. At all stages, the EFA cells adjacent to the ameloblasts showed CB-LI, however, some cells adjacent to the mature ameloblasts lacked the reaction. Intensive CB-LI was demonstrated in EFA cells at the reduced enamel epithelium. These immunohistochemical findings suggest EFA cells have cytochemical properties similar to those of ameloblasts.

Ultrastructural preservation of rat embryonic dental tissues after rapid fixation and dehydration under microwave irradiation.

Adequate preservation of the cells and matrix of mineralising tissues remains difficult, as organic components and initial mineral deposits may be lost during conventional processing for electron microscopy. In this study, we have reduced significantly the processing time using microwave irradiation. Rat molar tooth germs were fixed in 4% glutaraldehyde + 4% formaldehyde with 0.1 M sodium cacodylate in a laboratory microwave oven for two periods of 20 s with a maximal temperature of 37 degrees C. After conventional washing and post-fixation, specimens were dehydrated in graded ethanols under microwave irradiation for a total of 7 min 20 s. For comparison, some specimens were processed by conventional methods. After embedding, ultrathin sections were examined by electron microscopy. In differentiating ameloblasts and odontoblasts, plasma membranes, mitochondria, rough endoplasmic reticulum, the Golgi complex, together with all other cytoplasmic organelles exhibited excellent preservation. Microtubules, microfilaments and coated vesicles were particularly evident. Crystal-like mineral deposits were conspicuously present in relation to dentine matrix vesicles and collagen fibrils as well as in enamel matrix. The matrix of forming enamel had a globular electron-lucent appearance. It is concluded that this is a rapid method which provides a preserved or even improved morphology.

Subtilisin-like proprotein convertase PACE4 (SPC4) is a candidate processing enzyme of bone morphogenetic proteins during tooth formation.

The temporospatial expression of PACE4, a member of the mammalian subtilisin-like proprotein convertase family, in the developing rat molar tooth was determined by in situ hybridization. At the initiation stage of tooth development, PACE4 mRNA was weakly expressed in the dental lamina, whereas the mesenchymal cells intensely expressed the PACE4 transcript. At the bud stage, high-level expression of PACE4 mRNA was found in the dental epithelium and condensed dental mesenchyme. Its expression became more localized in the differentiating ameloblasts during cap and early bell stages. In the newborn rats, PACE4 mRNA was localized in the ameloblasts and odontoblasts, but its expression became weaker with advancing development, showing apparent association with the differentiation and establishment of functional ameloblasts and odontoblasts. These expression patterns of PACE4 were very similar to those of several bone morphogenetic proteins (BMPs) reported previously. Because BMPs, which are primarily involved in the morphogenesis in tooth formation, are synthesized as inactive precursors and activated by limited proteolysis at the consensus Arg-X-X-Arg maturation site, the present observations suggest that PACE4 is possibly a candidate proBMP convertase that acts during tooth formation.

Immunohistochemical localization of calbindin D28k during root formation of rat molar teeth.

The present study was undertaken to examine the localization of calbindin D28k (CB)-like immunoreactivity (-LI) during the root formation of the rat molar. In the adult rat, CB-LI was detected in some of the cells of the epithelial rest of Malassez at the bifurcational region and in certain cells between the root dentin and cementum at the apical region. These cells had indented nuclei and many tonofilaments, and cementocytes lacked CB-LI. Moreover, CB-LI was observed in the periodontal fibroblasts in the alveolar half of the apical region. During root formation, the cells in the Hertwig's epithelial root sheath (HERS) lacked CB-LI, but most fragmented cells along the root surface began to express CB-LI when HERS was disrupted. Preodontoblasts and odontoblasts at the apical portion of the root also showed CB-LI. After the formation of cellular cementum, the CB-immunoreactive (-IR) cells were entrapped between the root dentin and cementum in the apical portion of the root. The number of CB-IR cells at the root surface decreased gradually, while that between the root dentin and cementum increased. The fibroblasts in the periodontal ligament began to express CB-LI after commencement of the occlusion, and the number and the staining intensity of CB-IR fibroblasts increased gradually with the passage of time. The present results suggest that CB may play an important role in the survival of the epithelial cells, in the cellular responses of periodontal fibroblasts against mechanical forces caused by the occlusion, and in the initial mineralization by the odontoblasts through the regulation of intracellular Ca(2+) concentration.

Apoptosis in the early involuting stellate reticulum of rat molar tooth germs

When the enamel organ of the rat tooth germ is fully developed at the tip of the prospective cusp, amelogenesis begins, and at this site the overlaying stellate reticulum begins its involution. During the involution process, there is a gradual decrease in intercellular spaces, invasion by blood vessels, appearance of macrophage-like cells and reduction in the number of stellate reticulum cells. Since reduction or disappearance of cells during embryonic development in organs and tissues has been shown to occur by apoptosis, we decided to examine early involuting regions of the stellate reticulum in the hope of detecting apoptosis. For this purpose, upper first molars of Wistar newborn rats aged 1 and 3 days were fixed in formaldehyde for the TUNEL method and in glutaraldehyde-formaldehyde for light and electron microscopy. Paraffin sections revealed TUNEL-positive structures, i.e. brown-yellow-stained bodies, in the central portion of the stellate reticulum, and next to the outer enamel epithelium and stratum intermedium. Examination of ultrathin sections confirmed the TUNEL findings: some stellate reticulum cells showed nuclei containing crescent-like electron-opaque condensed masses of peripheral chromatin, typical of apoptosis. Also, apoptotic bodies of various sizes and appearances were frequently observed within stellate reticulum cells. We should like to suggest that apoptosis is associated with the reduction in the number of cells during regression of the reticulum.

Immunohistochemical localization of S-100β in the dental pulp of the rat molar

The present study was undertaken to reveal whether S-100α or S-100β or both are present in the nerve fibers in the rat molar tooth pulp. No immunoreactivity for S-100α was observed in the molar pulp. In the root pulp, thick smooth-surfaced structures accompanying the blood vessel showed S-100β-like immunoreactivity (-LI), and occasionally a very few thin beaded elements exhibited S-100β-LI. In the coronal pulp, S-100β-like immunoreactive (-IR) structures arborized repeatedly and extensively; they had a predominantly thick, smooth-surfaced appearance, though parts appeared thin and beaded. Numerous thin varicose S-100β-IR structures ran through the odontoblast cell layer, and further penetrated into the predentin alongside the dentinal tubules. They could be traced for approximately 10–20 μm into the predentin from the pulp-predentin border. Immunoelectron microscopy revealed that the Schwann cells in the root pulp showed S-100β-LI, and that S-100β-LI was present in the axoplasm as well as Schwann cells in the coronal pulp. The S-100β-IR axons were rarely surrounded by S-100β-IR Schwann cells. In the predentin, S-100β-IR nerve fibers terminated in a position close to the odontoblast processes. The present findings indicate that S-100β, not S-100α, is present in the axon in the dental pulp and predentin as well as in the Schwann cells.

Lectins inhibit periodontal ligament fibroblast attachment, spreading and migration on laminin substrates.

The ways in which carbohydrate signals from the extracellular matrix influence the responses of periodontal ligament (PDL) fibroblasts are essentially unknown. The purpose of this study, using video digital image analysis, was to examine the effects of lectins on the attachment, spreading and migrational responses of rat periodontal ligament fibroblasts on the highly glycosylated glycoprotein laminin (LN) in vitro. PDL fibroblasts were isolated from rat molar teeth and grown in culture. Bound LN and control substrates (bovine serum albumin and untreated plastic) were incubated with solutions of either wheatgerm agglutinin (WGA), specific for N-acetylglucosamine, or concanavalin A (ConA), specific for mannose, in 96-well plates. Unbound lectin was rinsed away and 10.0 x 10(3) cells were seeded per well and allowed to attach for 1.5 h. Incubation of LN substrates with WGA, prior to the addition of any cells, inhibited PDL fibroblast binding more than 5-fold. ConA, however, had no effect on cell binding but inhibited mean individual cell spreading nearly 3-fold under similar assay conditions. The effects could be prevented by adding each lectin's respective specific sugar. The lectins had no effects on the control substrates. In a 24-h cell migration assay WGA and ConA both significantly inhibited migration of PDL fibroblasts. It is likely that WGA inhibited cell attachment and cell migration, by binding to oligosaccharides and blocking access to adjacent polypeptide cell recognition sequences on LN. The results from the ConA experiments, where binding was allowed but spreading was severely inhibited, suggest a possible informational role for the carbohydrates present on LN.

Expression of Galpha proteins in the developing, denervated, or injured rat molar tooth.

The purpose of this study was to map the distribution of alpha-subunits of G-proteins--Galpha(olf s), Galpha(olf), Galpha(s), Galpha(i), Galpha(o), Galpha(z) and Galpha(q11)--in developing, denervated or injured rat molar teeth, using fluorescence microscopic immunohistochemistry coupled with immunogold electron microscopic immunocytochemistry. In rat fetuses (E17-E21), a widespread expression of Galpha(q11) was seen in maxillary/mandibular mesenchyme as well as in developing teeth. In addition, intensely Galpha(o)-positive nerve fibers were associated with the dental epithelium and the dental papilla of developing teeth. Other G proteins were absent or sparsely distributed during early tooth development. In the adult tooth pulp, odontoblasts appeared to express mainly Galpha(olf s), Galpha(o), and Galpha(q11). Nerve fibers were immunoreactive to Galpha(i), Galpha(o) and Galpha(z). In addition, pulpal blood vessels expressed varying levels of Galpha(olf s) Galpha(z) and Galpha(q11) while Galpha(olf s), Galpha(olf), Galpha(o) and Galpha(q11) were found in various pulpal mesenchymal cells. After adult denervation, nerve fiber-related G-protein immunoreactivity disappeared, but no other changes in pulpal G-protein immunoreactivity were noted. Odontoblasts and mesenchyme cells were intensely Galpha(i)-positive underneath a pulpal traumatic exposure, indicating an injury-induced pulpal upregulation of Galpha(i). The findings that Galpha(i), Galpha(o) and Galpha(z) are expressed in pulpal sensory nerve fibers suggest that these G proteins participate in signal conveyance from the target to the trigeminal nerve cell body.

Immunolocalisation of collagens in the developing rat molar tooth.

This study identifies different types of collagens during tooth development, maturation and ageing. Tissues from the rat first molar (from animals ranging in age from E14 to 104 wk postnatally) were immunostained using a panel of mono- and polyclonal antibodies against types I, II, III, IV, VI, IX and X collagen, fibronectin and laminin. During tooth development, types I and III collagens were expressed in the dental papilla at all stages but were also unexpectedly observed in the stellate reticulum of the enamel organ. Transient expression of type II collagen was also observed in the stellate reticulum during the late bell stage. Types IV and VI collagens, with laminin and fibronectin, were located within the basement membranes of the tooth germ. Collagen types I and III were observed within the developing follicle/periodontal ligament, type III predominating where collagen fibres were inserting into the alveolar bone and cementum. The pattern of types I and III collagen labelling within the periodontal ligament and the dental pulp did not change with age. Thus, some unusual collagen localisations were observed in the tooth germ, particularly within the stellate reticulum.

S100 protein-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

S100-immunoreactivity (ir) was examined in tooth pulp primary neurons of the rat. An immunofluorescence method demonstrated that the molar tooth pulp contained S100-immunoreactive (ir) nerve fibers. In the root pulp, pulp horn and roof of the pulp chamber, S100-ir smooth and varicose fibers ramified and formed subodontoblastic nerve plexuses. All the fibers became varicose at the base of the odontoblastic layer and extended to the odontoblastic layer. Some varicose endings could be traced into the dentin. The trigeminal neurons retrogradely labeled with fluorogold (FG) from the first and second maxillary molar tooth pulps exhibited S100- and parvalbumin-ir. Approximately 60% and 24% of the labeled cells were ir for S100 and parvalbumin, respectively. Virtually all parvalbumin-ir FG-labeled cells showed S100-ir, while 40% of S100-ir ones coexpressed parvalbumin-ir. An immunoelectron microscopic method revealed that all myelinated axons and half of the unmyelinated axons in the root pulp contained S100-ir. In the odontoblastic layer, predentin and dentin, S100-ir neurites lost the Schwann cell ensheathment and made close contact with cell bodies and processes of odontoblasts. The odontoblastic layer also contained parvalbumin-ir neurites. These neurites were devoid of the Schwann cell ensheathment and in close apposition to cell bodies and processes of odontoblasts. S100-ir pulpal axons seemed to be insensitive to repeated neonatal capsaicin treatment. This study suggests that S100-ir tooth pulp primary neurons are mostly myelinated and that S100-ir unmyelinated axons in the root pulp are preterminal segments of myelinated stem axons.

Calbindin D28K-like immunoreactive nerve fibres in the predentine of rat molar teeth

Immunoelectron-microscopy was applied to reveal the existence of nerve fibres and terminals showing calbindin D28k (CB)-like immunoreactivity (IR) in the rat molar tooth pulp. In the root pulp, thick, smooth-surfaced CB-IR nerve fibres were in bundles accompanying the blood vessels. In the coronal pulp, the fibres arborized repeatedly and extensively. CB-IR nerve fibres had a predominantly thick, smooth-surfaced appearance, though parts appeared thin and beaded. Occasionally some thin, varicose CB-IR nerve fibres ran along the odontoblasts, penetrating into the predentine alongside the dentinal tubules. They could be traced for approx. 10–20 μm into the predentine from the pulp-predentine border. Immunoelectron-microscopy revealed that only some of the nerve terminals in the predentine showed CB-IR, and that predentinal CB-IR nerve terminals were located close to the odontoblast processes. No synaptic structures were observed between them. The presence of CB-IR nerve terminals in the predentine suggests that many, if not all, CB-IR nerve fibres could be nociceptors. The CB could be involved in Ca 2+ homeostasis during the activation of nociceptors.

Involvement of nitric oxide in re-innervation of rat molar tooth pulp following transection of the inferior alveolar nerve

The aim of this study was to elucidate whether nitric oxide (NO) is involved in re-innervation of rat molar tooth pulp following transection of the inferior alveolar nerve. The inferior alveolar nerves (IAN) of rats were transected unilaterally under anesthesia with chloral hydrate. The animals received horseradish peroxidase (HRP) application to mandibular molar tooth pulps on both sides and were fixed by transvascular perfusion. The average number of labeled cells on each side of the trigeminal ganglion was not significantly different [101±11 (mean±S.E.M.; n=6, left) and 89±11 ( n=6, right)]. With HRP application on postoperative day 3, the ratio of the number of labeled neurons in the transected vs. non-transected (contralateral) sides was 31.5±5.8% ( n=11). The i.p. administration of N ω -nitro- l-arginine methyl ester ( l-NAME; 100 mg/kg, once a day for a period of 4 days), but not d-NAME, significantly decreased the ratio of the number of labeled neurons (10.1±7.0%, n=10). l-Arginine (300 mg/kg, i.p., once a day for a period of 4 days) slightly increased the number of labeled neurons on the transected side. Clonidine (25 μg/kg, i.p., once a day for a period of 4 days) failed to exhibit any significant effect on nerve regeneration. In the trigeminal ganglion ipsilateral to the transected IAN on postoperative day 4, NADPH-diaphorase (NADPH-d)-positive neurons had significantly increased. On the other hand, no changes in NADPH-d were observed in the superficial layers of the subnucleus caudalis of the spinal trigeminal nucleus from where primary neurons innervating the mammalian tooth pulp project. These results suggest that NO is involved in several mechanisms related to neuronal regeneration.