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.

Neurotrophin-4/5-depletion induces a delay in maturation of the periodontal Ruffini endings in mice.

Neurotrophin-4/5 (NT-4/5) - a member of the neurotrophic factors - is a ligand for TrkB, which has been reported to be expressed in the mechanoreceptive Ruffini endings of the periodontal ligament. The present study examined developmental changes in the terminal morphology and neural density in homozygous mice with a targeted disruption of the nt-4/5 gene and wild-type mice by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by quantitative analysis using an image analyzer. Postnatal development of terminal Schwann cells was also investigated by enzymatic histochemistry for non-specific cholinesterase activity (ChE). Furthermore, the immuno-expression of TrkB and low affinity nerve growth factor receptor (p75-NGFR) was surveyed in the periodontal Ruffini endings as well as trigeminal ganglion. At postnatal 1 week, the lingual periodontal ligament of both types of mice contained PGP 9.5-positive nerve fibers showing a tree-like ramification with axonal swellings in their course. In both types of mice at 2 weeks of age, comparatively thick nerve fibers with a smooth outline increased in number, and frequently ramified to form nerve terminals with dendritic profiles. However, no typical Ruffini endings with irregular outlines observed in the adult wild-type mice were found in the periodontal ligament at this stage. At postnatal 3 weeks, typical Ruffini endings with irregular outlines were discernable in the periodontal ligament of the wild-type mice while the dendritic endings showing smooth outlines were restricted to the homozygous mice. After postnatal 8 weeks, both types of mice showed an increase in the number of Ruffini endings, but the morphology differed between the wild-type and NT-4/5 homozygous mice. In the wild-type mice, a major population of the Ruffini endings expanded their axonal branches and developed many microprojections, resulting in a reduction of endings with smooth outlines. In contrast, we failed to find such typical Ruffini endings in the periodontal ligament of the homozygous mice: A majority of the periodontal Ruffini endings continued to show smooth outlines at postnatal 12 weeks. Quantitative analysis on neural density demonstrated a reduction in the homozygous mice with a significant difference by postnatal 8 weeks. Enzymatic histochemistry for non-specific ChE did not exhibit a distinct difference in the distribution and density of terminal Schwann cells between wild-type and homozygous mice. Furthermore, TrkB and p75-NGFR mRNA and proteins did not differ in the trigeminal ganglion between the two types. The periodontal Ruffini endings also displayed immunoreactivities for TrkB and p75- NGFR in both phenotypes. These findings suggest that the nt-4/5 gene depletion caused a delay in the formation and maturation of the periodontal Ruffini endings in the mice by inhibiting the growth of the periodontal nerves at an early stage, and indicate that multiple neurotrophins such as NT- 4/5 and BDNF might play roles in the development and/or maturation of the periodontal Ruffini endings.