Simultaneous visualization for coexpression of multiple neurotrophic factors in living Schwann cells

Schwann cells, as specialized glial cells found in the peripheral nervous system (PNS), produce a variety of neurotrophic factors (NTFs) and play a vital role in maintaining PNS functions. The combined biological effects of multiple NTFs are strongly associated with their coexpression characteristics in the physiological environment of living cells. In this study, the method for visualizing coexpression of multiple NTFs in living Schwann cells was investigated. We isolated Schwann cells from rat sciatic nerve and co-cultured them with dorsal root ganglion (DRG) neurons. The DRG neurons were removed, following which the Schwann cells were hybridized with fluorescence-labeled oligonucleotide probes for nerve growth factor (NGF), ciliary neurotrophic factor (CNTF) and glial cell line-derived neurotrophic factor (GDNF) introduced by electroporation and observed under confocal laser scanning (CLS) microscope. Our experimental results revealed that the following factors were crucial for visualizing the coexpression of multiple NTFs in living cells: (1) probe design and labeling, (2) probe specificity, (3) electroporation parameters for introducing probes into living cells and (4) hybridization signal detection. Our study may provide further insights into the synergistic effects of these factors on neurons.