| Purpose To determine the underlying mechanisms of neuroprotective effects the electrical stimulation (ES) exerts towards light-induced photoreceptor degeneration in vitro.
Methods After exposing cone photoreceptor-derived line (661w) cells cultivated in the plates to 5 hours’ bright blue light illumination, we cocultured them with primary retina microglia cells or Müller cells seeded in the transwell for 3, 6, 12, 24 hours precedingly receiving 1-hour ES or not. The mRNA and protein levels of inflammatory cytokines such as IL-1beta, TNF-alpha and neurotrophic factors such as BDNF, CNTF secreted from microglia or Müller cells at each time point were determined by ELISA, real-time PCR, Western blotting respectively. Meanwhile, LDH assay and TUNEL were used to evaluate cell death and apoptosis of 661w cells. The morphologic change of those cells were analysed using the immunocytochemistry. In the group of 24-hour time point, we also applied different regimens of ES to the transwell system to decide the optimal parameters triggering the maximum neuroprotective effects.
Results TUNEL and LDH assay showed that ES applied to the cocultured systems of light-reared 661w cells with microglia cells or Müller cells increased the survival of 661w cells, and the degree of rescue depended on the pulse duration and current intensity. ELISA, real-time PCR and Western blotting analyses revealed secretion of IL-1beta, TNF-alpha from microglia cells and BDNF, CNTF from Müller cells at a greater amount when those cells were cocultured with light-exposed 661w cells compared to the control group. Also we found gradual downregulation of IL-1beta, TNF-alpha and upregulation of BDNF, CNTF after receiving ES. At the same time, the antibody staining (Iba-1, ED1) showed a reduced rate of microglia cell activation after ES.
Conclusions These results demonstrate that electrical stimulation triggers antiapoptotic effects towards light-reared photoreceptor cells by suppressing microglia hyperactivity and enhancing Müller cells cell neuroprotective ability. The study also suggests that electrical stimulation can be a novel and potent therapeutic option to delay the progression of photoreceptor degeneration in patients suffering from retinitis pigmentosa. |
