Introduction:: Aging is the primary risk factor for cognitive decline. The impairment of neurovascular coupling, i.e., functional hyperemia, reported in numerous human and animal experimental studies is believed to contribute to the pathogenesis of dementia, including Alzheimer’s disease and vascular cognitive impairment, among other disorders. We and others recently showed that cerebral functional hyperemia is initiated in capillaries, thus unravelling one of the long-standing puzzles about the origin of functional hyperemia; transient increases in extracellular potassium (K+) and the initial dips in oxygen tension (PO2) together drive capillary hyperemia induced by sensory input. In addition, RBCs augment the well-known mechanisms of neurovascular coupling by acting as autonomous regulators of brain capillary perfusion. Extending upon this earlier work, we herein employed high-speed two-photon microscopy in vivo combined with recording and manipulation of PO2 and K+ to evaluate how aging affects the very initial cerebral hyperemia, i.e., capillary hyperemia, in mice. We supplemented the in vivo studies with an ex vivo oxygen consumption analysis in cortical tissues and microfluidic study of RBCs from young and aged mice. Thereby, we tested the linked hypotheses that the initiation of functional hyperemia is compromised in the aged mammalian brain due to (1) a suppression of cerebral O2 metabolism, (2) a failure of RBCs to increase their elasticity in response to reduced PO2, and (3) an age-dependent reduction of the O2 release rate by RBCs. All factors that will act to suppress activity-dependent increases in blood flow and O2 delivery in aging.
Materials and Methods:: Animals and surgical preparation. Young (2-5 months) were obtained from the Jackson Laboratory and old (20-23 months) wild type mice from the National Institute on Aging. A custom-made metal plate was glued to the skull and a 1.5-2.5 mm diameter cranial window was made over the hindlimb cortex for imaging (stereotaxic coordinates: 0.5–3 mm lateral; −1.5 to +1 mm anterior to bregma). Physiological manipulations. Hindlimb stimulation was delivered using 2 ms square-wave pulses at an intensity of 0.5-0.7 mA in a 10 Hz train lasting 2 s and captured with Clampex 9.0 software. Intrinsic optical signals were captured at 52 frames per second with a 12-bit INFINITY2-1M CCD camera. Two-photon imaging was performed using a custom-built microscope attached to a MaiTai HP Ti:Sapphire laser, and a 20 × objective. Local tissue O2 tension was recorded with a calibrated, modified Clark-type polarographic O2 microelectrode. Local K+ concentration was measured using a double-barreled K+-selective microelectrode. Microfluidic device and ex vivo measurement of RBC velocity. Microfluidic chips were fabricated with polydimethylsiloxane using standard soft-photolithographic techniques. To measure RBC capillary velocity, we used a microfluidic channel with a constriction of the 4.5 µm height (h), 3 µm width (wc) and 300 µm length (lc). Microfluidic devices were connected via a short polyethylene tube to an RBC reservoir, where a constant pressure (1.6 psi) was applied by a gas regulator with a precision of 0.1 psi. To create an O2 sink, sodium sulfite was added to a customized glass chamber containing the microfluidic device.
Results, Conclusions, and Discussions:: Upon sensory stimulation of the hindlimb in adult young and old wildtype mice, the magnitude and duration of evoked local field potential, the total ECoG power were not significantly different between groups. However, the increase of RBC capillary velocity in aged mice was suppressed, demonstrating compromised capillary hyperemia in the aged brain. Because local changes of PO2 contribute to capillary hyperemia, we examined stimulation-induced changes of PO2 in the activated hindlimb cortex and showed smaller and delayed PO2 dips in aged mice. Direct microinjection of oxygen scavengers to lower PO2 to a similar level between young and aged brain, however, the increase in capillary RBC velocity was still significantly lower in old mice, implying that RBCs in old mice respond less efficiently to local reductions in PO2. We thus used an ex vivo microfluidic setup to measure RBCs velocity response to low PO2 and showed that the sensitivity of RBC velocity to PO2 changes was significantly lower for old mice than for young mice. Discussions We showed that the initiation of cerebral functional hyperemia is compromised in aged murine brain. The mechanistic underpinning is multifaceted, but center around impaired O2 metabolism and delivery. We document that in the aged brain, the baseline PO2 has level is ~ 50% lower relative to young mice and transient PO2 dips in response both to neuronal activity and direct elevations of [K+]e are suppressed. Second, direct lowering of PO2 induced by microinjection of an oxygen scavenger was also less capable of increasing capillary RBC velocity in the aged brain. This in vivo observation was supplemented by microfluidic analysis showing that RBC velocity response to low PO2 were lower in aged than in young mice. Together, these observations document that aging is linked to fundamental changes in O2 delivery and metabolism that affect both resting PO2 and PO2 dips in responses to sensory input. Conclusions Our findings during functional activation concur in showing that reduced metabolic responsiveness of the aged brain and impaired reactions of RBCs to excessively low PO2 together contribute to the delayed initiation of functional hyperemia and hypoxia in the aged brain.