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Int. J. Mol. Sci. 2020, 21, 2370 14 of 20 9 min delivering 32.4 J/cm2 of energy. RL was produced by an LED device (Warp10, Quantum Devices; Barneveld, WI, USA) and NIRL by a diode laser with Slit Lamp Adapter and Continuous-Wave-Mode (OcuLight®SLx, Iridex; Mountain View, CA, USA)). The used organ culture provides cells in a vital and reactive status and exhibits the advantage of uniform and optimally controlled conditions for light application. 4.3. Intracellular ROS Production For evaluating ROS, we used CM-H2DCFDA (Molecular Probes®-Invitrogen, Carlsbad, CA, USA), an indicator of general ROS production in the form of hydrogen peroxide (H2O2), peroxynitrite anions (ONOO-), hydroxyl radicals (HO.), peroxide radicals (ROO.), superoxide (O ·−) or singlet oxygen (1O ). 22 After cultivation, retinas were dissected and stained with 25 μM CM-H2DCFDA for 10 min at 37 ◦C. Samples were rinsed in PBS and fixed in 6% PFA for 0.5 h. After embedding in 4% agarose, the explants were cut in 40 μm vertical vibratome sections. Retinas were immediately analyzed using a Zeiss LSM 510 confocal laser scanning microscope. Same acquisition settings were used in all experiments and groups. The mean fluorescence intensities of OS and IS were determined in eight regions of interest (ROI), normalized to Control. 4.4. Measurement of ATP Content The assessment of intracellular ATP content was performed utilizing ATP Bioluminescence Assay Kit HS II (Sigma-Aldrich; St. Louis, MI, USA) according to the manufacturer’s instructions and measured by a Tecan infinite M200 plate reader. 4.5. Histochemical Reactions for ETC I and II Activity ETC I and II enzyme activity was analyzed using unfixed 14 μm tick cryosections. To analyze NADH Coenzyme Q oxidoreductase (Complex I) activity, sections were incubated at 37 ◦C for 1 h with the following incubation medium: 2 mM NADH, 0.6 mM nitroblue tetrazolium chloride (NBT) in 0.1 M phosphate buffer, pH 7.4. For Succinic dehydrogenase (Complex II) histochemical assay, sections were incubated at 37 ◦C for 60 min with the following incubation medium: 50 mM succinic acid, 1.5 mM NBT; 5 mM EDTA, 1 mM sodium azide, 1 mM 1-Methoxy-5-methylphenazinium methyl sulfate (mPMS) in 0.1 M phosphate buffer, pH 7.6. Control sections were incubated in absence of substrate. To stop the reaction, slides were washed twice for 5 min with PBS, mounted with a coverslip and immediately analyzed using a Zeiss Axio Scope.A1 microscope. The same acquisition settings were used in all experiments and groups. The mean grey value of OS and IS was determined in 16 ROIs, normalized to Control. 4.6. Oxygraphic Measurements To measure the oxygen consumption from purified OS [71] from six–eight retina per group, an amperometric electrode (Unisense-Microsrespiration, Unisense A/S, Aarhus N, Denmark) was used. The experiment was performed in a closed chamber at 23 ◦C. Each sample (0.04 mg) was diluted 3:1 in ultrapure water, then added to the mixture by means of a Hamilton syringe, which granted a partial disruption of the OS, allowing substrates to permeate. Incubation medium was: 50 mM HEPES, 100 mM KCl, 2 mM MgCl2, 5 mM KH2PO4, 25 μg/mL ampicillin and 0.3 mM di(adenosine-5′) penta-phosphate as adenylate kinase inhibitor, pH 7.3 [72]. The content of the chamber was continuously mixed by an electromagnetic stirrer. Measurements were conducted under uncoupled conditions adding nigericin (5 μM) and valinomycin (10 μM) to the mixture prior to the sample additions. Oxidative substrates were: 20 mM fumarate to stimulate Complex I-III-IV pathway and 20 mM succinate to stimulate Complex II-III-IV pathway.PDF Image | Photobiomodulation Mediates Neuroprotection against Retinal
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