Finally, we compared light-induced blood flow changes in all four groups of mice. The animals were injected with SR101 and, after recording of basal blood flow in all vascular layers, were stimulated with a 30 second long 5Hz flickering blue light (480 nm;
Fig. 5A). This light exposure was estimated to be sufficient to directly stimulate both photoreceptors and ChR2 in cholinergic cells of ChAT-ChR2 mice. Indeed, in nondiabetic wt mice, a significant increase in blood flow was induced in the superficial (nondiabetic, 6 mice, 12 samples: basal 44 ± 2 cell/s and after stimulation 47 ± 2 cell/s; paired
t-test,
P < 0.001) and deep (nondiabetic, 6 mice, 12 samples: basal 44 ± 3 cell/s and after stimulation 47 ± 2 cell/s; paired
t-test,
P < 0.001) vascular layers. However, whereas the majority of capillaries in the intermediate vascular layer displayed an increased blood flow, only few capillaries had a reduction in their blood flow, thus eliminating the statistical significance of the change (nondiabetic, 6 mice, 12 samples: basal 42 ± 2 cell/s and after stimulation 44 ± 3 cell/s; paired
t-test,
P = 0.13; 50% capillaries increased blood flow). This bidirectional change of blood flow in response to the ChR2-activating light may be a physiological way to redistribute blood flow between the layers based on the neuronal demand. Similar trends were observed in nondiabetic ChAT-ChR2 mice in the superficial (nondiabetic ChAT-ChR2, 8 mice, 16 samples: basal 41 ± 3 cell/s and after stimulation 45 ± 4 cell/s; paired
t-test,
P < 0.001), intermediate (nondiabetic ChAT-ChR2, 8 mice, 24 samples: basal 43 ± 2 cell/s and after stimulation 42 ± 4 cell/s; paired
t-test,
P = 0.08; 33% capillaries increased blood flow), and deep (nondiabetic ChAT-ChR2, 8 mice, 16 samples: basal 41 ± 2 cell/s and after stimulation 45 ± 2 cell/s; paired
t-test,
P < 0.001) vascular layers. In contrast, no functional hyperemia was evident in diabetic nontransgenic mice with no detectable blood flow changes in the superficial (diabetic, 6 mice, 12 samples: basal 36 ± 3 cell/s and after stimulation 37 ± 3 cell/s; paired
t-test,
P = 0.3), intermediate (diabetic, 6 mice, 18 samples: 36 ± 4 cell/s; and after stimulation 36 ± 4 cell/s; paired
t-test,
P = 0.4), and deep (diabetic, 6 mice, 18 samples: 35 ± 5 cell/s; and after stimulation 36 ± 6 cell/s; paired
t-test,
P = 0.3) vascular layers. In diabetic ChAT-ChR2 mice, little change in blood flow was detected in the superficial (diabetic ChAT-ChR2, 8 mice, 16 samples: 41 ± 3 cell/s; and after stimulation 42 ± 3 cell/s; paired
t-test,
P = 0.023) but not in the deep layer (diabetic ChAT-ChR2, 8 mice, 24 samples: 41 ± 4 cell/s; and after stimulation 36 ± 6 cell/s; paired
t-test,
P = 0.4). In the intermediate layer, blood flow did not change (diabetic ChAT-ChR2, 8 mice, 16 samples: 41 ± 3 cell/s; and after stimulation 41 ± 4 cell/s; paired
t-test,
P = 0.42).