Spreading depolarizations (SDs) occur spontaneously in the cerebral cortex of subarachnoid hemorrhage, stroke or traumatic brain injury patients. Accumulating evidence prove that SDs exacerbate focal ischemic injury by converting zones of the viable but non-functional ischemic penumbra to the core region beyond rescue. Yet the SD-related mechanisms to mediate neurodegeneration remain poorly understood. Here we show in the cerebral cortex of isoflurane-anesthetized, young and old laboratory rats, that SDs propagating under ischemic penumbra-like conditions decrease intra and- extracellular tissue pH transiently to levels, which have been recognized to cause tissue damage. Further, tissue pH after the passage of each spontaneous SD event remains acidic for over 10 minutes. Finally, the recovery from SD-related tissue acidosis is hampered further by age. We propose that accumulating acid load is an effective mechanism for SD to cause delayed cell death in the ischemic nervous tissue, particularly in the aged brain.

Increasing evidence from epidemiological, clinical, and experimental studies indicates that cerebromicrovascular dysfunction and microcirculatory damage play critical roles in the pathogenesis of many types of dementia in the elderly, including both vascular cognitive impairment (VCI) and Alzheimer's disease. Vascular contributions to cognitive impairment and dementia (VCID) include impairment of neurovascular coupling responses/functional hyperemia (Bneurovascular uncoupling^). Due to the growing interest in understanding and pharmacologically targeting pathophysiological mechanisms of VCID, there is an increasing need for sensitive, easy-to-establish methods to assess neurovascular coupling responses. Laser speckle contrast imaging (LSCI) is a technique that allows rapid and minimally invasive visualization of changes in regional cerebromicrovascular blood perfusion. This type of imaging technique combines high resolution and speed to provide great spatiotemporal accuracy to measure moment-to-moment changes in cerebral blood flow induced by neuronal activation. Here, we provide detailed protocols for the successful measurement in neurovascular coupling responses in anesthetized mice equipped with a thinned-skull cranial window using LSCI. This method can be used to evaluate the effects of anti-aging or anti-AD treatments on cerebromicrovascular health.Keywords Neurovascular coupling . Functional hyperemia . Laser speckle contrast imaging . Laser speckle contrast analysis . LASCA . Laser speckle imaging . LSI Neurovascular uncoupling in aging and Alzheimer's diseaseIt is well recognized that the brain consumes more energy than any other human organ. Over 20% of the body's total energy requirements are spent to fuel the brain, which in turn only accounts for 2% of the total body mass. Moment-to-moment regulation of cerebral blood flow (CBF) is crucial since inadequate supply of glucose and oxygen to an active region of the brain GeroScience (2017) 39:465-473

Spreading depolarization (SD) events contribute to lesion maturation in the acutely injured human brain. Neurodegeneration related to SD is thought to be caused by the insufficiency of the cerebral blood flow (CBF) response; yet the mediators of the CBF response, or their deficiency in the aged or ischemic cerebral cortex, remain the target of intensive research. Here, we postulated that tissue pH effectively modulates the magnitude of hyperemia in response to SD, the coupling of which is prone to be dysfunctional in the aged or ischemic cerebral cortex. To test this hypothesis, we conducted systematic correlation analysis between the direct current (DC) potential signature of SD, SD-associated tissue acidosis, and hyperemic element of the CBF response in the isoflurane-anesthetized, young or old, and intact or ischemic rat cerebral cortex. The data demonstrate that the amplitude of the SD-related DC potential shift, tissue acidosis, and hyperemia are tightly coupled in the young intact cortex; ischemia and old age uncouples the amplitude of hyperemia from the amplitude of the DC potential shift and acidosis; the duration of the DC potential shift, hyperemia and acidosis positively correlate under ischemia alone; and old age disproportionally elongates the duration of acidosis with respect to the DC potential shift and hyperemia under ischemia. The coincidence of the variables supports the view that local CBF regulation with SD must have an effective metabolic component, which becomes dysfunctional with age or under ischemia. Finally, the known age-related acceleration of ischemic neurodegeneration may be promoted by exaggerated tissue acidosis. The hyperemic element of the cerebral blood flow response to spreading depolarization is effectively modulated by tissue pH in the young intact rat cerebral cortex. This coupling becomes dysfunctional with age or under ischemia, and tissue acidosis lasts disproportionally longer in the aged cortex, making the tissue increasingly more vulnerable.

Spreading depolarizations (SDs) indicate injury progression and predict worse clinical outcome in acute brain injury. We demonstrate in rodents that acute brain swelling upon cerebral ischemia impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic extracellular glutamate accumulation (>15 µM) predisposes an extensive bulk of tissue (4–5 mm2) for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices exposed to osmotic stress that SiD is the pathological expansion of prior punctual SD foci (0.5–1 mm2), is associated with astrocyte swelling, and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl− co-transporters, or volume-regulated anion channels mitigated slice edema, extracellular glutamate accumulation (<10 µM) and SiD occurrence. Reversal of slice swelling by hyperosmotic mannitol counteracted glutamate accumulation and prevented SiD. In contrast, inhibition of glial metabolism or inhibition of astrocyte glutamate transporters reproduced the SiD phenotype. Finally, we show in the rodent water intoxication model of cytotoxic edema that astrocyte swelling and altered astrocyte calcium waves are central in the evolution of SiD. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in acute brain injury.

Spreading depolarizations (SD) indicate infarct maturation and predict worse clinical outcome in ischemic stroke. We demonstrate here in rodents that brain edema formation upon ischemic stroke impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic glutamate accumulation predisposes an extensive bulk of tissue for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices under hypo-osmotic stress that SiD is the pathological expansion of prior SD foci, is associated with astrocyte swelling and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl- co-transporters, or volume-regulated anion channels mitigated slice edema, glutamate accumulation and SiD occurrence. Reversal of slice edema by hyperosmotic treatment counteracted glutamate accumulation and prevented SiD. In contrast, paralysis of astrocyte metabolism or inhibition of astrocyte glutamate uptake reproduced the SiD phenotype. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in ischemic stroke.