The results presented in this work demonstrate the dynamic effect of total acute ischemia on the autofluorescence spectra of in vivo
mouse skin keratinocytes. Linear spectral unmixing analysis suggests that the metabolically active fluorophores are protein-bound and free NADH. The contributions of protein-bound and free NADH show different dynamic reactions to hypoxia, anoxia and ischemia. NADH protein-bound to the complex I (NADH:ubiquinone oxidoreductase) of mitochondria is the major source of protein-bound NADH fluorescence. The temporal protein-bound NADH profile shows the dynamics of the transition of normoxia to hypoxia to anoxia. The ratio between protein-bound and free NADH change when the cells shift to anaerobic metabolism and finally the deprivation of glucose reduces the total NADH fluorescence. Our results, therefore, signify that in mouse keratinocytes all the major metabolic pathways (glycolysis, anaerobic fermentation and oxidative phosphorylation) respond to the loss of blood-supplied (via capillaries) oxygen and glucose. Another important implication of our results is the confirmation of the presence of metabolically functional mitochondria in epidermal keratinocytes, contrary to the conclusion of a recent study that keratinocytes are functionally anaerobic and that keratinocytic mitochondria are metabolically dysfunctional [27
]. Our observation showing significant response of mitochondrial-bound NADH to ischemia strongly suggests inhibition of mitochondrial complex I, hence, an evidence of mitochondrial oxidative phosphorylation of keratinocytes under normoxic conditions.
The results also demonstrate that although there is evidence supporting the hypothesis that cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of epidermis [28
], capillary oxygen supply remains to be the major source for epidermal metabolic function. Based on optical measurements of oxygen flux on the surface of human skin, measured values of oxygen diffusion in skin tissues, and assumption of homogeneity of skin, the thickness of the skin tissue that is supplied by atmospheric oxygen was estimated to be 266–375 μm [28
]. The measurements presented in the present study were carried out at depths of ~50 μm relative to the surface of the skin and the analysis of the results clearly showed metabolic response to complete deprivation of blood-supplied oxygen. On the other hand, the possibility that obstruction of blood supply establishes a hypoxic rather than an anoxic environment cannot be disregarded. In fact, it may even explain the slow metabolic response (~2 h) of the keratinocytes following the loss of blood-supplied oxygen.
The interpretation of the results of this study relies significantly on the validity of the spectral unmixing procedure which included fitting of the spectra with mathematically-modeled Gaussians as reference spectra. Although the Gaussian components do not exactly represent the spectra of pure compounds, the results strongly suggest that these fitted components are convenient representations of protein-bound and free NADH. On the other hand, the use of spectra of pure compounds as reference spectra in spectral unmixing will not yield more accurate results since the measured emission spectra are modulated by tissue inner filter effects (i
., light scattering and absorption in turbid media), tissue viscosity, refractive index and solvent effects [5
]. Furthermore, the experiments were necessarily carried out under anesthesia, and thus some level of anesthesia-related change particularly in the normoxic condition could influence the dynamic metabolic response of epidermal keratinocytes. A recent report showed a small change (<9%) in skin oxygenation of mice after one hour of administration of ketamine anesthesia under normoxic conditions [30
Most imaging experiments on free and protein-bound NADH were carried out using two-photon excited fluorescence lifetime imaging (FLIM) [8
]. Discrimination between the free and protein-bound NADH is achieved by taking advantage of the difference in their fluorescence lifetimes: the short lifetime component is attributed to the free NADH (τfreeNADH
= 0.3 to 0.5 ns) while the long lifetime component is attributed to the protein-bound NADH (τboundNADH
= 1.6 to 3.7 ns) [8
]. Comparison between FLIM and spectral imaging in terms of ability to discriminate between protein-bound and free NADH is not straightforward and depending on the type of experiment different aspects need to be taken into account. An important factor in the in-vivo imaging of NADH is the sensitivity and non-invasiveness of the applied method: autofluorescence levels are in general low and in in-vivo experiments only low excitation levels are tolerated. The high time resolution of FLIM dictates the use of photomultiplier tubes or avalanche photo diodes with peak quantum efficiencies in the range of 10-40% while the present spectral imaging setup is equipped with a detector with a >90% peak quantum efficiency. Moreover, FLIM requires the use of band-pass filters to select the emission of the NADH and suppress autofluorescence contributions from other components such as flavins. In contrast, spectral imaging detects a broad spectral range covering the entire (protein-bound and free) NADH emission band. In our experience both techniques require comparable amounts of signal for reliable analyses of the protein-bound and free NADH components. The higher sensitivity of spectral imaging results in discrimination between the two components at relatively short pixel dwell times of 2 ms per pixel compared to 11 to 17 ms per pixel [9
] in FLIM studies. Furthermore, the spectral imaging provides valuable additional information on morphology and the presence of other components besides NADH.
Finally, this study has demonstrated the capability of nonlinear spectral metabolic imaging in obtaining both morphological and biochemical information to unravel the dynamic metabolic response of living cells inside tissues for a long period of time following acute ischemia. Our results represent clear indication that spectral imaging effectively discriminates protein-bound and free NADH.