The ciliary zonules, also known as the zonules of Zinn, help to control the thickness of the lens during focusing. The ciliary zonules are composed of oxytalan fibers, which are synthesized by human nonpigmented ciliary epithelial cells (HNPCEC). The ciliary zonules are exposed to ultraviolet (UV), especially UV-A and UV-B, throughout life. We previously demonstrated that UV-B, but not UV-A, degrades fibrillin-1- and fibrillin-2-positive oxytalan fibers. However, the mechanism by which UV-B degrades oxytalan fibers remains unknown. In this study, we investigate the involvement of matrix metalloproteinase-2 (MMP-2) in the UV-B-induced degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs. Enzyme-linked immunosorbent assay revealed that UV-B irradiation at levels of 100 and 150 mJ/cm2 significantly increased the level of active MMP-2. Notably, MMP-2 inhibitors completely suppressed the degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers. In addition, we show that UV-B activates MMP-2 via stress-responsive kinase p38. Taken together, the results suggest that UV-B activates a production of active type of MMP-2 via the p38 pathway, and subsequently, an active-type MMP-2 degrades the fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs.
Proprioception from masticatory apparatus and periodontal ligaments comes through the trigeminal mesencephalic nucleus (Vmes). We evaluated the effects of tooth loss on neurodegeneration of the Vmes and trigeminal motor nucleus (Vmo). Bilateral maxillary molars of 2-month-old C57BL/6J mice were extracted under anesthesia. Neural projections of the Vmes to the periodontium were confirmed by injecting Fluoro-Gold (FG) retrogradely into the extraction sockets, and for the anterograde labeling adeno-associated virus encoding green fluorescent protein (AAV-GFP) was applied. For immunohistochemistry, Piezo2, ATF3, Caspase 3, ChAT and TDP-43 antibodies were used. At 1 month after tooth extraction, the number of Piezo2-immunoreactive (IR) Vmes neurons were decreased significantly. ATF3-IR neurons were detected on day 5 after tooth extraction. Dead cleaved caspase-3-IR neurons were found among Vmes neurons on days 7 and 12. In the Vmo, neuronal cytoplasmic inclusions (NCIs) formation type of TDP-43 increased at 1 and 2 months after extraction. These indicate the existence of neural projections from the Vmes to the periodontium in mice and that tooth loss induces the death of Vmes neurons followed by TDP-43 pathology in the Vmo. Therefore, tooth loss induces Vmes neuronal cell death, causing Vmo neurodegeneration and presumably affecting masticatory function.
Software based analyses of immunohistochemical staining are designed for obtaining quantitative, reproducible, and objective data. However, often times only a certain type of positive cells or structures need to be quantified thus whole image analysis cannot be performed. Such an example is Hofbauer placental cells, which show positivity of some antigens together with trophoblast, but only Hofbauer cells represent the regions of interest (ROIs). Two independent observers evaluated the immunohistochemical staining intensity of Hofbauer cells in placenta samples stained for cytoplasmic antigens by ImageJ, QuPath and light microscopy. Thus, the precise manual determination of ROIs, i.e. Hofbauer cells, was necessary. We detected low inter-observer variability in staining intensity. Almost perfect agreement between observers was reached for ImageJ and QuPath whilst substantial agreement was reached for light microscopy evaluation. As for the comparison of ImageJ, QuPath and light microscopy, the agreement of all three methods (identical immunohistochemical intensity) was achieved for 38.1% samples. The almost perfect agreement of staining intensities was reached between ImageJ and QuPath, and moderate agreement for comparison of the light microscopy to both software. Software analyses are much more time-consuming, thus their utilization is at least questionable to evaluate ROIs with selection.