Fumiko Harada

Detection of acid-sensing ion channel 3 (ASIC3) in periodontal Ruffini endings of mouse incisors.

The acid-sensing ion channel 3 (ASIC3), a member of the epithelial sodium channel/degenerin (ENaC/DEG) superfamily, has been reported to participate in acid sensing, mechanosensation, and nociception. However, no information is available regarding the precise localization and function of this molecule in the periodontal ligament, which contains abundant sensory nerves originating from the trigeminal ganglion. The present study examined the expression of ASIC3 in the lingual periodontal ligament of mouse incisors by immunohistochemistry. Furthermore, the expression of ASIC3 in the trigeminal ganglion - which innervates the periodontal ligament - was investigated at protein (immunohistochemistry and quantitative analysis) and mRNA levels (RT-PCR technique and in situ hybridization histochemistry). Immunohistochemistry for ASIC3 was able to demonstrate dendritic profiles of the periodontal Ruffini endings in the mouse incisors. No thin fibers terminating as nociceptive free nerve endings exhibited ASIC3 immunoreactivity. Double immunofluorescent staining revealed ASIC3 immunoreaction in the axoplasm but not in the ordinary Schwann cells - including the associated terminal Schwann cells. Observation of the trigeminal ganglia showed variously sized neurons expressing ASIC3 immunoreaction; the most intense immunopositivity was found in the small and medium-sized neurons, as confirmed by in situ hybridization histochemistry using a specific cRNA probe. Quantitative analysis on trigeminal ganglion neurons showed that 38.0% of ASIC3 neurons could be categorized as medium-sized neurons which mediate mechanotransduction. These findings suggest that ASIC3 functions as a molecule for mechanosensation in the periodontal Ruffini endings.

Involvement of neurotrophin-4/5 in regeneration of the periodontal Ruffini endings at the early stage

Little is known about the role of neurotrophin‐4/5 (NT‐4/5) in the regeneration of mechanoreceptors. Therefore, the present study examined the regeneration process of Ruffini endings in the periodontal ligament in nt‐4/5‐deficient and wildtype mice following transection of the inferior alveolar nerve by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by computer‐assisted quantitative image analysis. Furthermore, rescue experiments by a continuous administration of recombinant NT‐4/5 were performed and analyzed quantitatively. At postoperative day 3 (PO 3d), almost all PGP 9.5‐positive neural elements had disappeared; they began to appear in both types of animals at PO 7d. At PO 10d, almost all nerve fibers showed a beaded appearance, with fewer ramifications in both types of mice. Although the regeneration proceeded in the wildtype, a major population of the periodontal Ruffini endings continued to display smooth outlines at PO 28d in the nt‐4/5 homozygous mice. The reduction ratio of neural density reached a maximum at PO 3d, decreased at PO 10d, and later showed a plateau. In a rescue experiment, an administration of NT‐4/5 showed an acceleration of nerve regeneration in the homozygous mice. These findings indicate that the nt‐4/5‐depletion causes a delay in the regeneration of the periodontal Ruffini endings, but the delay is shortened by an exogenous administration of NT‐4/5. Combined with our previous findings of bdnf‐deficient mice (Harada et al. [ 2003 ] Arch Histol Cytol 66:183–194), these morphological and numerical data suggest that multiple neurotrophins such as NT‐4/5 and brain‐derived neurotrophic factor (BDNF) play roles in their regeneration in a stage‐specific manner. J. Comp. Neurol. 501:400–412, 2007.

Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve.

The present study examined the immunohistochemical localization of heat shock protein 25 (Hsp25) during the regeneration of nerve fibers and Schwann cells in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve. In the untreated control group, the periodontal ligament of rat incisor did not contain any Hsp25-immunoreaction. On postoperative day 3 (PO 3d), a small number of Schwann cells with slender cytoplasmic processes exhibited Hsp25-immunoreactivity. From PO 5d to PO 21d, Hsp25-positive nerve fibers and Schwann cells drastically increased in number in the alveolar half of the ligament. Although the axons of some regenerating Ruffini-like endings also showed Hsp25-immunoreactions, the migrated Schwann cells were devoid of Hsp25-immunoreaction. Thereafter, Hsp25-positive structures decreased in number gradually to disappear from the periodontal ligament by PO 56d. This temporal expression of Hsp25 in the periodontal ligament well-reflected the regeneration process of the nerve fibers. Hsp25 in the regenerating nerve fibers and denervated Schwann cells most likely serves in modulating actin dynamics and as a cellular inhibitor of apoptosis, respectively.

GDNF Expression in Terminal Schwann Cells Associated With the Periodontal Ruffini Endings of the Rat Incisors During Nerve Regeneration

The terminal Schwann cells (TSCs) which play crucial roles in regeneration of the periodontal Ruffini endings (RE) exhibit immunoreaction for glial cell line‐derived neurotrophic factor (GDNF). However, no information is available regarding the role of GDNF in the periodontal RE during nerve regeneration. This study was undertaken to examine the changes in GDNF expression in the rat periodontal RE following transection of the inferior alveolar nerve (IAN) using immunohistochemistry for GDNF and S‐100 protein, a marker for the TSCs. We additionally investigated the changes in expression of GDNF in the trigeminal ganglion (TG) at protein and mRNA levels. A transection to IAN induced a disappearance of the TSCs from the alveolus‐related part (ARP), followed by a migration of spindle‐shaped cells with S‐100 but without GDNF immunoreactions into the tooth‐related part (TRP) by postoperative (PO) week 2. At PO week 2, GDNF immunoreacted cellular elements increased in number in the ARP although the spindle‐shaped cells without GDNF reaction remained in the TRP. After PO week 4, many GDNF‐positive TSCs appeared in the ARP though the spindle‐shaped cells vanished from the TRP. A real time RT‐PCR analysis demonstrated the highest elevation of GDNF mRNA in the TG at PO week 2. These findings suggested the involvement of this molecule in the maturation and maintenance of the periodontal RE during regeneration. Taken together with our previous and current studies, it appears that the regeneration of the periodontal RE is controlled by multiple neurotrophins in a stage‐specific manner. Anat Rec, 2009.

Occurrence of two β-tubulin isoforms with different polymerizing abilities in L5178Y cells

Mouse lymphoma L5178Y cells express at least two isoforms of beta-tubulin, designated M beta I and M beta II, as revealed by isoelectrofocusing, whereas two independently isolated normal T-cell clones, 3D10 and K23, express only M beta I. M beta II-tubulin is more acidic (pI, 5.10) than M beta I-tubulin (pI, 5.15). L5178Y cells were disrupted under the microtubule-stabilizing conditions, followed by centrifugation to separate fractions containing polymerized and unpolymerized tubulin. We found that a proportion of M beta II to total beta-tubulins is larger in the fraction containing unpolymerized tubulin than in that containing polymerized tubulin. In addition, when tubulin was purified from extracts of L5178Y cells by repeated cycles of polymerization-depolymerization, the M beta II-tubulin isoform was gradually lost during the successive purification steps. The low recovery of M beta II-tubulin was observed, irrespective of the presence or absence of MAPs, and even in the presence of an excess amount of essentially polymerizable porcine brain tubulin. These results indicate that M beta II-tubulin is less able to polymerize than is M beta I-tubulin, both in vivo and in vitro.

Contribution of synovial lining cells to synovial vascularization of the rat temporomandibular joint.

The lining layer of the synovial membrane in the temporomandibular joint (TMJ) contains two types of lining cells: macrophage‐like type A and fibroblast‐like type B cells. The type B cells are particularly heterogeneous in their morphology and immunoreactivity, so that details of their functions remain unclear. Some of the type B cells exhibit certain resemblances in their ultrastructure to those of an activated capillary pericyte at the initial stage of the angiogenesis. The articular surface, composed of cartilage and the disc in the TMJ, has few vasculatures, whereas the synovial lining layer is richly equipped with blood capillaries to produce the constituent of synovial fluid. The present study investigated at both the light and electron microscopic levels the immunocytochemical characteristics of the synovial lining cells in the adult rat TMJ, focusing on their contribution to the synovial vascularization. It also employed an intravascular perfusion with Lycopersicon esculentum (tomato) lectin to identify functional vessels in vivo. Results showed that several type B cells expressed desmin, a muscle‐specific intermediate filament which is known as the earliest protein to appear during myogenesis as well as being a marker for the immature capillary pericyte. These desmin‐positive type B cells showed immunoreactions for vimentin and pericyte markers (neuron‐glial 2; NG2 and PDGFRβ) but not for the other markers of myogenic cells (MyoD and myogenin) or a contractile apparatus (αSMA and caldesmon). Immunoreactivity for RECA‐1, an endothelial marker, was observed in the macrophage‐like type A cells. The arterioles and venules inside the synovial folds extended numerous capillaries with RECA‐1‐positive endothelial cells and desmin‐positive pericytes to distribute densely in the lining layer. The distal portion of these capillaries showing RECA‐1‐immunoreactivity lacked lectin‐staining, indicating a loss of blood‐circulation due to sprouting or termination in the lining layer. The desmin‐positive type B and RECA‐1‐positive type A cells attached to this portion of the capillaries. Some capillaries in the lining layer also expressed ninein, a marker for sprouting endothelial cells, called tip cells. Since an activated pericyte, macrophage and tip cell are known to act together at the forefront of the vessel sprout during angiogenesis, the desmin‐positive type B cell and RECA‐1‐positive type A cell might serve as these angiogenic cells in the synovial lining layer. Tomato lectin perfusion following decalcification would be a highly useful tool for research on the vasculature of the mineralized tissue. Use of this technique combined with immunohistochemistry should permit future extensive investigations on the presence of the physiological angiogenesis and on the function of the lining cells in the synovial membrane.

Nonsurgical treatment of adult skeletal Class III malocclusion with crowding and missing four premolars corrected with extraction of mandibular first molars

Abstract An adult male patient was present with anterior cross bite. He was referred to Orthodontic Clinic of Niigata University Medical and Dental Hospital for recommendation of surgical orthodontic treatment by general practitioner. The patient was diagnosed as skeletal Class III with anterior crowding and missing four premolars. To correct anterior cross bite and crowding, surgical orthodontic treatment was considered, but the patient refused orthognathic surgery. We therefore determined that nonsurgical treatment with mandibular bilateral first molar extraction would be indicated. After treatment, suitable overjet, overbite and proper functional occlusion were attained with Class I molar relation. Active treatment was 34 months, and the treatment result remained stable 2 years and 4 months after debonding.