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. 2018 Sep 13;11:333. doi: 10.3389/fnmol.2018.00333

Figure 4.

Figure 4

AMPA provokes oligodendroglial damage in isolated optic nerves from PLP-DsRed transgenic mice. Isolated optic nerves from PLP-DsRed transgenic mice were preincubated in artificial CSF medium in absence or presence of TBB (25 μM; 3 h) or SP600125 (20 μM; 1 h) and subjected to excitotoxic insult by stimulation of AMPA receptors (100 μM CTZ plus 100 μM AMPA) during 2 h. (A,B) Lactate dehydrogenase (LDH) release quantification at 30, 60 and 90 min post-stimulus showed that TBB or SP600125 pre-treated optic nerves exhibited a significant reduction in LDH release at all post-stimulus times analyzed. **p < 0.005; ***p < 0.0001 (optic nerves treated with AMPA vs optic nerves control); #p < 0.05; ##p < 0.01; ###p < 0.001 (vs. optic nerves treated only with AMPA). (C,D) Representative fields of z-stacks of optic nerves from PLP-DsRed transgenic mice control or stimulated with AMPA alone (C) or in presence of inhibitors TBB or SP600125 (D). Note a loss of oligodendrocytes in optic nerves exposed to AMPA, which was prevented by incubation of agonist in presence of TBB or SP600125. (E) Quantification of the fluorescence signal emitted by oligodendrocytes of optic nerves from P25 PLP-DsRed mice, expressed as arbitrary units of fluorescence. ***p < 0.001 (optic nerves treated with AMPA vs. optic nerves control); ##p < 0.01, ###p < 0.001 (vs. optic nerves treated only with AMPA). (F) Cell counts of PLP+ oligodendrocytes were performed and data were represented as mean number of cells (±SEM, n > 4 animals) in a constant volume (FOV), as detailed in “Materials and Methods” section. ***p < 0.001 (optic nerves treated with AMPA vs. optic nerves control); #p < 0.05, ###p < 0.001 (vs. optic nerves treated only with AMPA). Both quantifications show that oligodendrocyte loss induced by AMPA excitotoxic insult was inhibited by the presence of TBB or SP600125 inhibitors. (G) Optic nerves from PLP-DsRed transgenic mice were processed for immunofluorescence using antibodies to APC and nuclei were stained with DAPI. Treatment with AMPA induced a dramatic fluorescence loss in both PLP and APC oligodendroglial markers, indicating severe oligodendrocyte damage. In addition, DAPI labeling revealed nuclei condenzation in AMPA-treated optic nerves (arrows). Scale bar 20 μm.