| Part One:Neuroprotective effect of electrical stimulation promoting light-injured photoreceptor cell survivalPhotoreceptor cell line (661W) in culture was seeded on6-well plate for24h. Then the adherent661W cells were exposed to15,000lux intense blue light for4～6h in an incubator.24h after light exposure, we used LDH, TUNEL assay and immunocytochemistry to detect the cell death, apoptosis and morphological changes of661W cells. The results showed that the cell death rate of661W was59.7%±2.7%analyzed by LDH assay, and there was70%TUNEL positive apoptotic661W cells24h after light exposure. Meanwhile, the immunostaining showed that light-exposed661W cells incubated alone for24h had become spindle-shaped and appeared to have larger intracellular spaces than the normal661W cells, which were bigger and flattened. The above results indicated the success of establishing the light-induced photoreceptor degeneration model.1h biphasic, direct electrical stimulation (ES,20Hz,300～1600μA) was administered in advance to the co-culture of light-damaged661W cells and microglia or Muller cells (treatment group). The co-culture without receiving ES was considered as untreated group, while the co-culture of normal661W cells and microglia or Muller cells was considered as normal control. LDH assay was conducted3h,6h,12h and24h post co-culture respectively. TUNEL assay and immunocytochemistry were used24h post co-culture. The LDH apoptosis-time curve showed that treating the co-culture of light-injured661W cells and microglia with1000μA ES significantly decreased the cell death rate from12hpc (12h post co-culture) and remained almost unchanged until24hpl relative to untreated group. While administration of1600μA ES to the co-culture of light-injured661W cells and Miiller cells decreased the cell death rate from3hpl and was attenuated to the lowest at24hpl compared with the untreated group. TUNEL assay indicated that there were obviously fewer TUNEL positive cells in the treatment group than those in untreated group. The immunostaining results showed that when administered1-h ES before co-incubation, this co-culture showed relatively milder morphological changes after24h relative to no treatment. On the other hand, we also applied ES on light-damaged661W cells cultivated alone. However, contrary to the results in the co-culture systems,1h ES induced no significant attenuation effect on cell death of those light-damaged661W cells.Hence these results imply that ES is able to ameliorate intense light-induced photoreceptor degeneration. However, photoreceptor cells themselves are not responsive to ES. Actually, it is possible that ES has changed the microenvironment surrounding photoreceptors such as glia cells activity, while the alteration of glia cells activity in turn influences the survival of photoreceptor cells.Part Two:Light-induced photoreceptor degeneration activates glia cells, and activated glia cells have impact on light-damaged photoreceptor apoptosisAfter establishment of light-induced photoreceptor degeneration in vitro model, we further took advantage of the co-culture of glia cells and light-damaged photoreceptor cells (untreated group) to explore the proliferative, morphological and accompanied functional change of microglia or Muller cells influenced by light-injured photoreceptor cells, also to observe the parallel survival state change of661W cells. The co-culture of normal661W cells and microglia or Muller cells was considered the normal control. At24hpc, the immunocytochemistry was used to detect the morphological and numerical difference of microglia or Muller cells between untreated group and normal control. Real-time PCR and Western blot were used to analyze the mRNA transcription and protein expression difference between two groups of IL-1β, TNF-α secretion from microglia and BDNF, CNTF production from Muller cells respectively at3hpc,6hpc,12hpc,24hpc. The LDH, TUNEL assay and immunostaining method were used to observe the difference of cell death rate and apoptotic rate of661W cells between the group of light-injured661W cells cultivated alone and the co-culture of light-injured661W cells and microglia or Muller cells.The immunocytochemistry found that in the co-culture of microglia and normal661W cells (normal control), most of the cells were adherent, showing ramified appearance with long and thin processes. But a high proportion of microglia cultured with light-reared661W cells (untreated group) for24h exhibited ameboid morphology similar to that of monocytes and macrophages with larger amounts than those in normal control. There was no obvious morphological difference between the two groups of Muller cells co-culture. In addition, at24hpc, real time PCR and Western blot analysis showed that IL-1β and TNF-α mRNA expression and protein synthesis was significantly upregulated in microglia cultured with light-reared661W cells relative to microglia cultured with normal661W cells. Meanwhile, BDNF and CNTF mRNA expression and protein synthesis in Muller cells cultured with light-exposed661W cells was also significantly increased compared with Muller cells cultured with normal661W cells. The timing curve of IL-1β and TNF-α protein expression levels showed that in normal control group, IL-1β and TNF-α protein expression levels remained stable and relatively low over time. On the contrary, the production of IL-1β protein in microglia cultured with light-reared661W cells gradually increased and peaked at24hpc. TNF-α production was also upregulated over time, reaching a peak at12hpc and declining a bit until24hpc. On the other hand, the time course in different co-cultures confirmed that BDNF protein expression in normal control became attenuated over time and CNTF protein expression was maintained at the same level. The level of BDNF in Muller cells cultured with light-damaged661W cells rapidly increased and peaked at6hpc but returned to baseline at24hpc, while CNTF expression in this co-culture rose placidly from3hpc to24hpc. The LDH and TUNEL assay observed that the cell death rate of light-injured661W cells cultured with microglia was significantly upregulated over time relative to that of light-damaged661W cells cultivated alone, while the cell death rate of light-damaged661W cells cultured with Muller cells was significantly decreased compared with that of light-damaged661W cells incubated alone.The results showed above indicate that light-induced photoreceptor degeneration triggers microglia activation and Muller cell reactive gliosis, accompanied with upregulation of proinflammatory cytokines1L-1α,TNF-α secretion from microglia and neurotrophic factors BDNF、CNTF production from Muller cells. Moreover, microglia activation deteriorates the extent of photoreceptor degeneration while Muller cell reactive gliosis ameliorates the extent of photoreceptor apoptosis. Thus, change of glia cell activated status play a role in the process of light-induced photoreceptor degeneration.Part Three:The interference effect of electrical stimulation on light-damaged photoreceptor induced glia cell activationOn the basis of establishing light-damaged photoreceptor cell model and co-culture system, ES was applied to the co-cultures in advance. Then the immunocytochemistry method was used to detect the morphological changes of microglia and Muller cells between each group (normal control, untreated group, treated group). Real-time PCR and Western blot were used to analyze the mRNA transcription and protein expression difference between each group of IL-1β, TNF-α secretion from microglia and BDNF, CNTF production from Muller cells respectively at3hpc,6hpc,12hpc,24hpc.The immunocytochemistry results of microglia and Muller cells in normal control and untreated group were mentioned in Part Two. However, in ES treated group, there was a decreased rate of microglial transformation to ameboid phagocytic status. Though lacking the long processes like those of "resting" cells incubated with normal661W cells, a lot of these ES-treated microglia cells in this co-culture maintained flat bodies. We defined this as intermediate status. Moreover, treated with ES, the soma of some Muller cells appeared larger and the number of cells increased in ES treated group. Along with morphological changes, in ES treated group, there was a prominent downregulation of IL-1β and TNF-α mRNA and protein levels in microglia relative to untreated group, while a further upregulation of BDNF and CNTF mRNA and protein levels in Muller cells was observed in ES treated group compared with untreated group. In addition, the protein synthesis time curve showed that following1-h ES before co-incubation, IL-1β and TNF-α protein expression in microglia fell to their lowest levels at24hpc and12hpc, respectively in treated group. However, treatment with ES further augmented the magnitude of BDNF upregulation in Muller cells at6hpc in treated group and declined a little until24hpc. Administration of ES also accelerated the time at which CNTF protein expression in Muller cells peaked at6hpc and further enhanced the increasing degree in this co-culture then sustained elevated until24hpc.The results determine that ES can provide neuroprotection for light-reared photoreceptors via the following two mechanisms:1) by inhibiting the activation of microglia cells to suppress their cytoxicity that mediates photoreceptor degeneration,2) by strengthening the reactive gliosis of Miiller cells to enhance their neurotrophic potential that ameliorates photoreceptor damage. Thus, ES is further determined to be a novel and potent therapeutic option to delay the progression of photoreceptor degeneration. |
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