Endoscopic confocal laser-microscopy for the intraoperative nerve recognition: is it feasible?

Surgeons lose most of their tactile tissue information during minimal invasive surgery and need an additional tool of intraoperative tissue recognition. Confocal laser microscopy (CLM) is a well-established method of tissue investigation. The objective of this study was to analyze the feasibility and diagnostic accuracy of CLM nervous tissue recognition. Images taken with an endoscopic CLM system of sympathetic ganglions, nerve fibers and pleural tissue were characterized in terms of specific signal-patterns ex-vivo. No fluorescent dye was used. Diagnostic accuracy of tissue classification was evaluated by newly trained observers (sensitivity, specificity, PPV, NPV and interobserver variability). Although CLM images showed low CLM image contrast, assessment of nerve tissue was feasible without any fluorescent dye. Sensitivity and specificity ranged between 0.73 and 0.9 and 0.55–1.0, respectively. PPVs were 0.71–1.0 and the NPV range was between 0.58 and 0.86. The overall interobserver variability was 0.36. The eCLM enables to evaluate nervous tissue and to distinguish between nerve fibers, ganglions and pleural tissue based on backscattered light. However, the low image contrast and the heterogeneity in correct tissue diagnosis and a fair interobserver variability indicate the limit of CLM imaging without any fluorescent dye.

Suppressive Effects of Hainosan (Painongsan) against Biofilm Production by Streptococcus mutans

Streptococcus mutans, a bacterium that causes dental plaques, forms a biofilm on tooth surfaces. This biofilm can cause gingivitis by stimulating the gingival margin. However, there is no established treatment for biofilm removal. Hainosan (Painongsan), a traditional Japanese Kampo formula, has been used to treat gingivitis. Therefore, we investigated the biofilm suppressive effects of the hainosan extract (HNS) and its components on S. mutans. We conducted scanning electron microscopy and confocal laser microscopy analyses to clarify the anti-biofilm activities of HNS and its crude drugs. We also performed a quantitative RT-PCR assay to assess the biofilm-related gene expression. HNS showed a significant dose-dependent suppressive effect on biofilm formation. Both the scanning electron microscopy and confocal laser microscopy analyses also revealed the significant inhibitory effects of the extract on biofilm formation. Transmission electron microscopy analysis showed that HNS disrupted the surface of the bacterial wall. Furthermore, HNS reduced the hydrophobicity of the bacteria, and suppressed the mRNA expression of β-glucosyltransferase (gtfB), glucosyltransferase-SI (gtfC), and fructosyltransferase (ftf). Among the constituents of hainosan, the extract of the root of Platycodon grandiflorum (PG) showed the strongest biofilm suppression effect. Platycodin D, one of the constituent natural compounds of PG, inhibited S. mutans-associated biofilm. These findings indicate that hainosan eliminates dental plaques by suppressing biofilm formation by S. mutans.

Casticin Induces DNA Damage and Affects DNA Repair Associated Protein Expression in Human Lung Cancer A549 Cells

Casticin was obtained from natural plants, and it has been shown to exert biological functions; however, no report concerns the induction of DNA damage and repair in human lung cancer cells. The objective of this study was to investigate the effects and molecular mechanism of casticin on DNA damage and repair in human lung cancer A549 cells. Cell viability was determined by flow cytometric assay. The DNA damage was evaluated by 4’,6-diamidino-2-phenylindole (DAPI) staining and electrophoresis which included comet assay and DNA gel electrophoresis. The protein levels associated with DNA damage and repair were analyzed by western blotting. The expression and translocation of p-H2A.X were observed by confocal laser microscopy. Casticin reduced total viable cell number and induced DNA condensation, fragmentation, and damage in A549 cells. Furthermore, casticin increased p-ATM at 6 h and increased p-ATR and BRCA1 at 6–24 h treatment but decreased p-ATM at 24–48 h, as well as decreased p-ATR and BRCA1 at 48 h. Furthermore, casticin decreased p-p53 at 6–24 h but increased at 48 h. Casticin increased p-H2A.X and MDC1 at 6–48 h treatment. In addition, casticin increased PARP (cleavage) at 6, 24, and 48 h treatment, DNA-PKcs and MGMT at 48 h in A549 cells. Casticin induced the expressions and nuclear translocation of p-H2AX in A549 cells by confocal laser microscopy. Casticin reduced cell number through DNA damage and condensation in human lung cancer A549 cells.