Basal nitric oxide production is enhanced by hydraulic pressure in cultured human periodontal ligament fibroblasts.

To understand orthodontic tooth movement and determine optimal orthodontic force from a biological viewpoint, nitric oxide production in cultured human periodontal ligament fibroblasts was measured at varying levels of hydraulic pressure. The fibroblasts in a culture flask were exposed to the controlled change in hydraulic pressure, and intracellular nitric oxide levels were measured in real time by a nitric oxide-binding fluorescent dye, diaminofluorescein-2. The fibroblasts produced a significantly larger amount of nitric oxide at the pressure of 75 and 100 mmHg, compared with the pressure of 0, 25, and 50 mmHg (P <.0001, one-way ANOVA, and P <.05, Tukey-Kramer test). Immunohistochemically, the cultured fibroblasts expressed brain nitric oxide synthase. The pressure level to enhance nitric oxide production was comparable to the magnitude of clinically used orthodontic force (80 g/cm(2)). Nitric oxide might be a key regulator in orthodontic tooth movement.

Calbindin-D28k-immunoreactivity in the trigeminal ganglion neurons and molar tooth pulp of the rat.

The cell body size and coexpression of carbonic anhydrase (CA), calretinin (CR) and calcitonin gene-related peptide (CGRP) of primary neurons with calbindin-D28k (CB) was examined in the trigeminal ganglion (TG) of the rat. CB-immunoreactive (-ir) cells were mostly large and preferentially distributed in the maxillary and mandibular divisions of the TG. 48% of CB-ir TG cells exhibited enzyme CA activity. 10% of CB-ir TG cells contained CR-ir. Most TG cells coexpressing CB- and CR-irs were localized to the maxillary and mandibular divisions and exhibited CA activity. 6.5% of CB-ir TG cells coexisted with CGRP-ir. 46% of TG cells coexpressing CB and CGRP exhibited CA activity. The innervation of the molar tooth pulp by CB-ir TG primary neurons was also examined. CB-ir thick and smooth nerve fibers projected from the root pulp to the pulp horn and the roof of the pulp chamber, where they became thinner and rarely entered the subodontoblastic layer. However, they could not be traced to the odontoblastic layer, predentin or dentine. The distribution pattern of CB-ir pulpal fibers was different from that of CR-ir ones. The trigeminal neurons cells retrogradely labeled with fast blue (FB) from the maxillary molar tooth pulp contained CB- and CR-irs. 23% and 1% of the labeled cells were immunoreactive for CB and CR, respectively. The coexpression of CB- and CR-immunoreactivities (-irs) in FB-labeled cells was negligible. An immunoelectron microscopic method revealed that 21% of pulpal nerve fibers were immunoreactive for CB, and that all CB-ir nerve fibers in the root pulp were myelinated. The present study indicated that the tooth pulp primary neurons contained CB-ir but did not coexpress CB- and CR-irs and that these neurons projected their myelinated axons to the pulp.

Intracellular calcium response to hydraulic pressure in human periodontal ligament fibroblasts.

Human periodontal ligament fibroblasts in culture were exposed to the controlled change in hydraulic pressure and were monitored continuously with an electric pressure gauge, and the concentration of intracellular calcium was measured in real time by a calcium-binding fluorescent dye, fluo-3. The elevation of hydraulic pressure to a level ranging from 20 to 50 mm Hg induced transient elevation of the intracellular calcium concentration in about 10% of the fibroblasts observed, indicating that these cells could respond to the pressure change. The results supported further an idea that periodontal ligament fibroblasts, responding to the pressure exerted by orthodontic force, would initiate the chain of events in orthodontic tooth movement, including alveolar bone remodeling. The threshold level of pressure (27 to 68 g/cm2) obtained in this experiment, at which the fibroblasts started to respond, would provide a biochemical basis to determine the optimal magnitude of stress for clinical orthodontics.

Parvalbumin- and calretinin-immunoreactive trigeminal neurons innervating the rat molar tooth pulp.

Calcium-binding proteins and neuropeptides were examined in trigeminal neuronal cell bodies retrogradely labeled with Fast blue (FB) from the maxillary molar tooth pulp of the rat. FB-labeled cells were located in the maxillary division of the trigeminal ganglion. Approximately 30 and 50% of the labeled cells were immunoreactive for parvalbumin and calcitonin gene-related peptide (CGRP), respectively. The coexpression of these substances was observed in 9.5% of FB-labeled cells. On the other hand, 2.4% of FB-labeled cells exhibited calretinin-immunoreactivity (CR-ir) and 20% tachykinin (TK)-ir. The coexpression of CR and TK was observed in 1.9% of FB-labeled cells, i.e., most of CR-ir FB-labeled neurons coexpressed TK-ir. An immuno-EM method revealed that all parvalbumin-ir nerve fibers in the root pulp were myelinated and that CGRP-ir nerve fibers were both myelinated (15%) and unmyelinated (85%). The present study indicated that primary nociceptors innervating the rat molar tooth pulp contained parvalbumin and CR and coexpressed these calcium-binding proteins and neuropeptides. It was suggested that peripheral axons of parvalbumin-ir tooth pulp primary neurons are all myelinated. Most peripheral CR-ir axons are probably unmyelinated because TK-ir myelinated axons have never been demonstrated in any peripheral organ.

Parvalbumin, calretinin and carbonic anhydrase in the trigeminal and spinal primary neurons of the rat.

The cell-body size of parvalbumin-immunoreactive (-ir) primary neurons was measured in the trigeminal (TG) and lumber dorsal root ganglia (DRG). In the DRG, parvalbumin-ir was mostly detected in large cells (94% in the range of 600-2800 microns2). Parvalbumin-ir TG cells were smaller than similar DRG cells and yet parvalbumin-ir TG cells of < 400 microns2 (2.86%) were rare. Trichrome stains for parvalbumin, calretinin (CR) and carbonic anhydrase (CA), and for parvalbumin, calcitonin gene-related peptide (CGRP) and CA were performed to estimate possible overlap of these substances. Virtually all parvalbumin-ir DRG cells contained CA activity while a small subpopulation (28.5%) of CR-ir DRG cells lacked CA activity. All the CR-ir DRG cells that exhibited CA were also ir for parvalbumin. 31.1% of parvalbumin-ir DRG cells exhibited CR-ir while 71.5% of CR-ir DRG cells showed parvalbumin-ir. All the CR-ir DRG cells of < 400 microns2 lacked CA activity and parvalbumin-ir while all those of > 800 microns2 exhibited both activities. Approximately 30% of CR-ir DRG cells in the size range of 400-800 microns2 co-expressed CA. DRG cells co-expressing parvalbumin and CGRP were rare (approximately 1%). As was the case for the DRG, most of parvalbumin-ir TG cells exhibited CA activity (89.24%) and lacked CGRP-ir (96.6%). CR-ir TG cells were also subdivided into two groups; one with and the other without co-expression of CA. Unlike in the DRG, however, co-expression of parvalbumin and CR could never be detected in the TG.

Neural parvalbumin and calretinin in the tooth pulp.

Parvalbumin- and calretinin-immunoreactivities (CR-irs) were examined in the molar tooth pulp of the rat using immunohistochemical methods. CR-ir fibers were further classified based on the tachykinin-ir revealed by a double immunofluorescence method. The rat root pulp contained three types of nerve fibers; parvalbumin-ir smooth fibers, CR-ir (TK-negative) smooth fibers and CR-ir (TK-ir) varicose fibers. These fibers projected toward the roof of the pulp chamber and pulp horn without marked ramification. In the subodontoblastic layer at the roof of the pulp chamber and pulp horn, parvalbumin-ir smooth fibers repeatedly ramified and extended varicose terminals into the odontoblastic layer. CR-ir (TK-negative) smooth fibers reached the subodontoblastic layer without marked ramification and gave rise to varicose terminals that appeared to terminate within the subodontoblastic layer. On the other hand, CR-ir (TK-ir) varicose fibers proceeded to the subodontoblastic layer at the roof of the pulp chamber and pulp horn, where they ramified and penetrated the odontoblastic layer. The present study indicates that the rat tooth pulp contains myelinated parvalbumin-ir and CR-ir (TK-negative) fibers, and unmyelinated CR-ir (TK-ir) fibers, and that they project varicose terminals to the subodontoblastic and odontoblastic layers. The central projection sites of these sensory fibers have yet to be revealed.

Changes in c-fos induction in dorsal horn neurons by hindpaw formalin stimulation following tibial neurotomy.

The hindpaw was partially denervated by the tibial nerve transection in adult rats. At post-transection intervals varying from 2 to 168 days, the hindpaw was stimulated bilaterally by subcutaneous injection of formalin. The excitability of dorsal horn neurons was expressed as the percentage ratio of the number of formalin-induced c-fos protein-like immunoreactive neurons (fos-neurons) on the neurotomized (experimental) side to that on the un-neurotomized (control) side. At 2 days post-injury, a marked reduction in the number of fos-neurons was noted in laminae I-VII of the lumbar spinal cord. Among these, reduction was greatest in the medial 3/8 of laminae I and II (terminal field of the tibial nerve, i.e. tibial territory), and smallest in the lateral 5/8 of the same laminae (the peroneal/hip territory). The low level of c-fos induction remained unchanged for 7 days. At 14 days, the excitability of neurons in all laminae showed a marked increase compared to the post-injury days 2 and 3 combined. Thereafter, the increased level of excitability in the tibial territory was maintained throughout the post-injury period examined in this study. On the other hand, a statistically significant increase in excitability in the peroneal/hip territory was only seen between 14 and 28 days and the excitability almost returned to the baseline (days 2 and 3 post-transection combined) level at 42 days. Although deeper laminae (III-VII) contained much less formalin-induced fos-neurons, they also exhibited post-injury excitability changes with a temporal pattern similar to that of the peroneal/hip territory of laminae I and II.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the tooth mobility change during the orthodontic tooth movement studied by means of Periotest and MIMD (the mechanical impedance measuring device for the periodontal tissue).

Two mechanical devices were selected to attempt to assess any changes in tooth mobility when orthodontic force was applied. A Periotest (PT) for assessing mobility and a mechanical impedance measuring device (MIMD) were the instruments chosen to diagnose the changes in the periodontal condition. The relative mobility of four canines after orthodontic tooth movement manipulation was assessed over a 4-week period. Both devices were able to detect very small changes in tooth mobility and could follow the changes during tooth movement. According to the Periotest unit, the tooth mobility changes were not within the clinically abnormal range, despite the initial retraction load from a sectional arch, applied in a horizontal direction with about 150 gm for each tooth. In all instances, the contiguous maxillary first premolar had been removed 1 to 2 months before force application. The tooth mobility observed in this investigation could be different from that caused by periodontal disease or traumatic injury. Just after orthodontic tooth movement manipulation, the Periotest values (PTV), as measured on the Periotest, of tooth mobility decreased and the resistance as measured by the mechanical impedance measuring device (MIMDM) was increased. Thus, initially, there is a greater resistance to movement with decreased mobility. After the experimental period of 4 weeks, there was an increase in the mobility, as measured by the Periotest and the mechanical resistance decrements were observed. However, the Periotest units showed some different changes occasionally that did not seem to reflect the state of tooth movement. This needs to be investigated further over a longer period of time. On the basis of the results obtained, it does appear that it is feasible to use these devices to determine mobility changes in patients at various stages of orthodontic treatment.

Calretinin-immunoreactivity in trigeminal neurons innervating the nasal mucosa of the rat.

Trigeminal primary neuronal cell bodies were labeled by retrograde transport of Fluoro-gold (FG) from the nasal mucosa of rats. The trigeminal ganglion containing the labeled cell bodies were processed for double stain for calretinin- and tachykinin-immunoreactivities (CR- and TK-irs). Except for a few contralateral cells, all the cells that innervated the nasal mucosa (NM cells) were confined to the ophthalmo-maxillary division of the trigeminal ganglion ipsilateral to the FG application. In the dorsal two-thirds of the ganglion, NM cells formed a cluster in the rostromedial part of ophthalmo-maxillary division (the rostromedial cluster). In the ventral third, the number of cells in the rostromedial cluster markedly decreased. Instead, numerous NM cells were found in the caudolateral part of the ophthalmo-maxillary division (the caudoventrolateral cluster). CR- and TK-irs were detected in 18% and 54% of overall population of NM cells, respectively. Virtually all of CR-immunoreactive (-ir) NM cells coexpressed TK. Although the proportion of TK-ir cells, irrespective of CR-ir, was similar for both clusters, CR-ir cells were more frequent in the caudoventrolateral cluster than in the rostromedial cluster. In the dorsal 1/3 of the ganglion where all the NM cells belonged to the rostromedial cluster, only 8.4% exhibited CR-ir. On the other hand, as much as 30.1% of NM cells expressed CR-ir in the ventral 1/3 where most NM cells were found in the caudoventrolateral cluster. Trigeminal cell bodies innervating the cornea and conjunctivum were located in the rostromedial part of the ophthalmo-maxillary division.(ABSTRACT TRUNCATED AT 250 WORDS)

c-Fos expression by dorsal horn neurons chronically deafferented by peripheral nerve section in response to spared, somatotopically inappropriate nociceptive primary input.

Subcutaneous formalin injection into the hindpaw of rats induces c-Fos expression in neurons in the ipsilateral spinal cord dorsal horn. In laminae I and II of the dorsal horn at the junction of 4th and 5th segments of the lumbar spinal cord, neurons exhibiting c-Fos protein-like immunoreactivity (Fos-LI) are concentrated in the medial 3/4 that correspond to the terminal field of primary neurons innervating the sciatic nerve. Subacute tibial nerve section 24 h before formalin stimulation caused almost complete elimination of neurons with the formalin-induced Fos-LI in the medial 1/2 (tibial territory) of the above sciatic territory of the dorsal horn. Following a longer survival period (chronic tibial nerve section of 21 days standing), neurons with the formalin-induced Fos-LI re-appeared in the tibial territory. In addition, the number of neurons with the formalin-induced Fos-LI increased in the medial part of the peroneal territory (the lateral 1/2 of the sciatic territory). The results indicate that the activation of c-Fos expression in that part of dorsal horn that has been chronically deafferented by the tibial nerve section is taken over by the spared, but somatotopically inappropriate primary nociceptors. Furthermore, dorsal horn neurons outside but near the deafferented tibial nerve's territory exhibit hypersensitivity to c-Fos expression evoked by intact, somatotopically appropriate primary nociceptive input.

[High construction bite activator treatment of anterior crossbite in mixed dentition].

We reported two cases of functional Class III malocclusion with a skeletal tendency in mixed dentition, treated by applying an activator with a high construction bite beyond the freeway space. A high construction bite, approximately 8 to 10 mm between upper and lower incisor edges was employed. By increasing the vertical separation of the construction bite to a specific range, the high construction bite intensified the activation of the myotatic reflex in the muscles of mastication as well the passive tension in the stretched perioral muscles through the viscoelastic properties. (Furthermore, the tension which was generated passively from the stretched perioral soft tissues, such as tendon and skin, would join the force). In these two cases, we could efficiently use the active and passive tension of the muscles by using an activator of this type, and gain the effective orthodontic force clinically.