Three-dimensional Analysis of Iba1+ Macrophages in Human Dental Pulp Using Whole Mount Immunostaining

Objectives: Macrophages and dendritic cells (DC) play an important role in the immune response and immunotolerance. Ionized calcium binding adaptor molecule 1 (Iba1) was identified as a 17,000 kDa calcium-binding protein that is specifically expressed by macrophages/microglia in the brain. The present report describes how we used Iba1 as a target for investigation of macrophage dynamics, and investigated relationships between Iba1+ macrophages and nerves three-dimensionally (3D) using whole mount immunostaining.
Methods: We retrieved the dental pulp tissue from a human extracted teeth (Tokyo Medical and Dental University Ethics Review Committee Approval No. 948). The tissue were fixed in 4% PFA. For immunohistochemistry, anti-Iba1, Factor XIIIa and anti-neurofilament antibodies were used as primary antibodies. 3D image construction was performed using a TCS SP8 (Leica).
Results: Several neurofilament+ nerve fibers in the root pulp ran parallel to the tooth axis, but the fibers showed a more complex divergence in the crown pulp. Iba1+ cells were mainly branched shape and were somewhat sparsely present in the root pulp where the majority were parallel to the tooth axis. These cells were densely present in the crown pulp, however, and no tendencies were noted with regard to cell directionality. There are large numbers of Factor XIIIa+ cells present in human dental pulp, and several of these are reported to be DC like cells. Here we used Iba1, widely used as a macrophage marker, to investigate the dynamics of Iba1+ cells in human dental pulp, and found that the cells had localization and morphological characteristics that resembled those of Factor XIIIa+ cells by 3D using whole mount immunostaining.
Conclusions: Large numbers of Iba1+ macrophages are present in human dental pulp and the distribution is similar to that of FactorXIIIa+ cells.