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. 2013 Jul;33(7):996-9.
doi: 10.1038/jcbfm.2013.63. Epub 2013 Apr 24.

In vivo NADH fluorescence imaging indicates effect of aquaporin-4 deletion on oxygen microdistribution in cortical spreading depression

Alexander S Thrane  1 , Takahiro Takano, Vinita Rangroo Thrane, Fushun Wang, Weiguo Peng, Ole Petter Ottersen, Maiken Nedergaard, Erlend A Nagelhus
Affiliations

In vivo NADH fluorescence imaging indicates effect of aquaporin-4 deletion on oxygen microdistribution in cortical spreading depression

Alexander S Thrane et al. J Cereb Blood Flow Metab. 2013 Jul.

Abstract

Using in vivo two-photon imaging, we show that mice deficient in aquaporin-4 (AQP4) display increased fluorescence of nicotinamide adenine dinucleotide (NADH) when subjected to cortical spreading depression. The increased NADH signal, a proxy of tissue hypoxia, was restricted to microwatershed areas remote from the vasculature. Aqp4 deletion had no effects on the hyperemia response, but slowed [K(+)]o recovery. These observations suggest that K(+) uptake is suppressed in Aqp4(-/-) mice as a consequence of decreased oxygen delivery to tissue located furthest away from the vascular source of oxygen, although increased oxygen consumption may also contribute to our observations.

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Figures

Figure 1
Figure 1
Deletion of aquaporin-4 (Aqp4) increased nicotinamide adenine dinucleotide (NADH) fluorescence in microwatershed areas during cortical spreading depression (CSD). (A) Experimental setup. After induction of CSD in living Aqp4−/− and wild-type (WT) mice, we measured changes in NADH fluorescence with 2PLSM, cerebral blood flow with laser Doppler flowmetry, extracellular concentration of K+ ([K+]o) with K+-ion-sensitive microelectrodes, direct current potential, and tissue partial pressure of oxygen (tpO2) (not illustrated). (B) Immunofluorescence micrographs from WT and Aqp4−/− mice illustrating AQP4 expression in cerebral cortex. AQP4 (green), GFAP (red), and DAPI (blue). (C) Endogenous NAD+ and its reduced counterpart NADH are key coenzymes in glycolysis, the citric acid cycle, and the mitochondrial respiratory chain. Since only NADH is fluorescent (and not NAD+), two-photon NADH imaging offers maps of tissue redox state that can be used as a proxy of tissue oxygenation. (D) Left: NADH fluorescence images of cerebral cortex in WT (75 μm depth) and Aqp4−/− (105 μm depth) mice. Green denotes fluorescein isothiocyanate-dextran labeled blood vessels. Nicotinamide adenine dinucleotide fluorescence is largely uniform, except where NADH is made less visible by the presence of large pial vessels at the surface of the brain. Right: Representative false color images of change in NADH fluorescence (ΔF/F0) from baseline intensity, during the early (CSD wave) and late phase of CSD (hypoxic phase), and after recovery. In the early phase of CSD, when the DC potential dropped, cortical tissue showed a complex pattern of NADH decrease (dip, shown in green) and increase (overshoot, shown in red/orange). At a later stage, when DC potential had normalized, all regions showed increased NADH fluorescence (hypoxic phase), before gradually returning to baseline level. Scale bar represents 50 μm. (E) Running average traces (ΔF/F0) for the biphasic NADH response during CSD, showing whole field (‘total'), dip and overshoot in WT and Aqp4−/− mice. Duration of DC shift is indicated. *P<0.05, n=five animals in each group, unpaired t test. (F) Bar graph representing the peak change in NADH fluorescence in WT and Aqp4−/− mice. P=0.394 (total), 0.329 (dip), and 0.00762 (overshoot), n=five animals in each group, unpaired t test. Data are shown as mean±s.e.m.
Figure 2
Figure 2
Deletion of aquaporin-4 (Aqp4) did not affect vascular oxygen supply but delayed extracellular K+ clearance in cortical spreading depression (CSD). (A) Representative laser Doppler recordings (black) and a running average of these recordings (red) demonstrating the hyperemic response during CSD in Aqp4−/− and WT mice. (B, C) Bar graphs summarizing the effect of Aqp4 deletion on peak amplitude and duration of the CSD hyperemic response. P=0.876 (peak), 0.794 (duration), n=22 wild-type (WT) and 41 Aqp4−/− stimulations from seven WT and 12 Aqp4−/− animals, unpaired t test. (D) Line graph showing mean cortical tpO2 before (basal) and during CSD in WT and Aqp4−/− mice, measured using oxygen-sensitive microelectrodes. P=0.264 (base), 0.555 (trough), n=6 WT and eight Aqp4−/− animals, unpaired t test. (E, F) Bar graphs showing mean cortical tpO2 slope decline and recovery. P=0.641 (decline), P=0.818 (recovery), n=6 WT and eight Aqp4−/− animals, unpaired t test. (G, H) Representative traces and bar graphs summarizing the effects of Aqp4 deletion on amplitude of the DC-shift. *P=0.0223, n=6 (WT) and eight (Aqp4−/−) animals, unpaired t test. (I) Running averages of extracellular concentration of K+([K+]o) during CSD for WT (black) and Aqp4−/− mice (red), acquired using K+-ion-sensitive-microelectrodes. Inset: K+-ISM calibration curve. n=7 (WT) and 12 (Aqp4−/−). (J-M) Bar graphs summarizing the effect of Aqp4 deletion on peak [K+]o, time to peak, [K+]o recovery rate, and duration of [K+]o elevation in CSD. [K+]o peak is reduced and [K+]o recovery is delayed in Aqp4−/− mice. *P=0.0459 (duration), **P<0.001 (peak and recovery rate), P=0.855 (time to peak), n=22 (WT) and 41 (Aqp4−/−) CSD responses from seven WT and 12 (Aqp4−/−) animals, unpaired t test. Data are shown as mean±s.e.m.
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