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Takeshi Morimoto, Hiroyuki Kanda, Takao Endo, Kohji Nishida, Takashi Fujikado; Watching dying RGC-5 cells by using Raman microscopy 2. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5854.
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© ARVO (1962-2015); The Authors (2016-present)
Label-free imaging is desirable for elucidating morphological and biochemical changes of neurons in vitro and in vivo. Raman microscopy provides high chemical contrast without requiring preprocessing or fluorescence staining of samples. Last year, we demonstrated dynamic imaging of molecular distribution in unstained RGC-5 cells by using Raman microscopy. In the present study, we analyzed raman spectra of cytochrome c (cyt c) obtained from RGC-5 cells to compare the level of cyt c protein and found a discrepancy between the decrease of intensity of raman spectra of cyt c and the decrease of the level of cyt c protein in RGC-5 cells.
Our originally developed slit-scanning Raman microscopy was used in this study. All the hyperspectral Raman scattering images were obtained with 532 nm excitation from a frequency-doubled Nd: YVO4 laser. Immortalized retinal ganglion cells (RGC-5) were used as a sample in all experiments. Prior to Raman imaging, the cells were seeded on a crystal and cultured. For Raman imaging, the culture was maintained in a Hepes-buffered Tyrode’s solution and then was exposed to 400 mM glutamate. Raman images were taken before and after administration of glutamate at 0, 30, 60, 120 minutes. The intensity of Raman spectra of cyt c at 740-750 cm-1 obtained from RGC-5 cells at each time points was measured and analyzed. Cyt c protein obtained from other RGC-5 cell cultures were also measured by ELISA immunoassay to compare the intensity of Raman spectra of cyt c.
The mean intensity of Raman cyt c in RGC-5 cells without glutamate was 9.58 ± 2.8 arbitrary unit (a.u.), the mean intensity of Raman cyt c considerably decreased after administration of glutamate, 120 minutes after administration of glutamate, the intensity of cyt c was very weak, 0.16 ± 0.08 a.u. (one-way ANOVA, P< 0.005). On the other hand, the level of cyt c protein in RGC-5 cells also significantly decreased after administration of glutamate (one-way ANOVA, P< 0.03). However, the mean concentration of cyt c protein in RGC-5 cells 120 min after adminstration was 8.47±4.6 ng/ml, which was 61.4 % of the value in RGC-5 cells without glutamate (13.8 ± 2.1 ng/ml).
The intensity of Raman cyt c indicted the stage of the death process of RGCs. But there was a discrepancy between the decrease of intensity of Raman cyt c and the the decrease of cyt c protein in RGCs. Further investigation is needed to elucidate this discrepancy.
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