Astrocytic network alterations have been reported in Alzheimer's disease (AD), but the underlying pathways have remained undefined. Here we measure astrocytic calcium, cerebral blood flow and amyloid-β plaques in vivo in a mouse model of AD using multiphoton microscopy. We find that astrocytic hyperactivity, consisting of single-cell transients and calcium waves, is most pronounced in reactive astrogliosis around plaques and is sometimes associated with local blood flow changes. We show that astroglial hyperactivity is reduced after P2 purinoreceptor blockade or nucleotide release through connexin hemichannels, but is augmented by increasing cortical ADP concentration. P2X receptor blockade has no effect, but inhibition of P2Y1 receptors, which are strongly expressed by reactive astrocytes surrounding plaques, completely normalizes astrocytic hyperactivity. Our data suggest that astroglial network dysfunction is mediated by purinergic signalling in reactive astrocytes, and that intervention aimed at P2Y1 receptors or hemichannel-mediated nucleotide release may help ameliorate network dysfunction in AD.

In rats, cortical spreading hyperaemia is coupled to a spreading neuroglial depolarization wave (spreading depression) under physiological conditions, whereas cortical spreading ischaemia is coupled to it if red blood cell products are present in the subarachnoid space. Spreading ischaemia has been proposed as the pathophysiological correlate of the widespread cortical infarcts abundantly found in autopsy studies of patients with subarachnoid haemorrhage. The purpose of the present study was to investigate whether the extracellular ion changes associated with the depolarization wave may cause the vasoconstriction underlying spreading ischaemia. We induced spreading ischaemia in vivo with the nitric oxide (NO) scavenger oxyhaemoglobin and an elevated K+ concentration in the subarachnoid space while slow potential, pH, extracellular volume and concentrations of K+, Na+, Ca2+ and Cl- were measured in the cortex with microelectrodes. We then extraluminally applied an ionic cocktail (cocktail(SI)) to the isolated middle cerebral artery in vitro, matching the ionic composition of the extracellular space as measured during spreading ischaemia in vivo. Extraluminal application of cocktail(SI) caused middle cerebral artery dilatation in the absence and constriction in the presence of NO synthase inhibition in vitro, corresponding with the occurrence of spreading hyperaemia in the presence and spreading ischaemia in the absence of NO in vivo. The L-type Ca2+ inhibitor nimodipine caused the cocktail(SI)-induced vasoconstriction to revert to vasodilatation in the absence of NO in vitro similar to the reversal of spreading ischaemia to spreading hyperaemia in response to nimodipine in vivo. We found that K+ was the predominant vasoconstrictor contained in cocktail(SI). Its vasoconstrictor action was augmented by NO synthase inhibition. Our results suggest that, under elevated baseline K+ as a hallmark of any condition of energy deficiency, the extracellular ion changes represent the essential mediator of the vascular response to spreading neuroglial depolarization. In the presence of NO they mediate vasodilatation and in its absence they mediate constriction.

Lesion volume measurements with magnetic resonance imaging are widely used to assess outcome in rodent models of stroke. In this study, we improved a mathematical framework to correct lesion size for edema which is based on manual delineation of the lesion and hemispheres. Furthermore, a novel MATLAB toolbox to register mouse brain MR images to the Allen brain atlas is presented. Its capability to calculate edema-corrected lesion size was compared to the manual approach. Automated image registration performed equally well in in a mouse middle cerebral artery occlusion model (Pearson r ¼ 0.976, p ¼ 2.265e-11). Information encapsulated in the registration was used to generate maps of edema induced tissue volume changes. These showed discrepancies to simplified tissue models underlying the manual approach. The presented techniques provide biologically more meaningful, voxel-wise biomarkers of vasogenic edema after stroke.

Recently, a universal, simple, and fail-safe mechanism has been proposed by which cerebral blood flow (CBF) might be coupled to oxygen metabolism during neuronal activation without the need for any tissue-based mechanism. According to this concept, vasodilation occurs by local erythrocytic release of nitric oxide or ATP wherever and whenever hemoglobin is deoxygenated, directly matching oxygen demand and supply in every tissue. For neurovascular coupling in the brain, we present experimental evidence challenging this view by applying an experimental regime operating without deoxy-hemoglobin. Hyperbaric hyperoxygenation (HBO) allowed us to prevent hemoglobin deoxygenation, as the oxygen that was physically dissolved in the tissue was sufficient to support oxidative metabolism. Regional CBF and regional cerebral blood oxygenation were measured using a cranial window preparation in anesthetized rats. Hemodynamic and neuronal responses to electrical forepaw stimulation or cortical spreading depression (CSD) were analyzed under normobaric normoxia and during HBO up to 4 ATA (standard atmospheres absolute). Inconsistent with the proposed mechanism, during HBO, CBF responses to functional activation or CSD were unchanged. Our results show that activation-induced CBF regulation in the brain does not operate through the release of vasoactive mediators on hemoglobin deoxygenation or through a tissue-based oxygen-sensing mechanism.

In rats, spreading depolarization induces vasodilation/hyperemia in naïve tissue but the inverse response when artificial cerebrospinal fluid is topically applied to the brain containing (a) a nitric oxide-lowering agent and (b) elevated K þ . The inverse response is characterized by severe vasoconstriction/ischemia. The perfusion deficit runs together with the depolarization in the tissue (¼spreading ischemia). Here, we found in male Wistar rats that pre-treatment with artificial cerebrospinal fluid containing elevated K þ in vivo led to a selective decline in a 2 /a 3 Na þ /K þ -ATPase activity, determined spectrophotometrically ex vivo. Moreover, spreading ischemia, recorded with laser-Doppler flowmetry and electrocorticography, resulted from artificial cerebrospinal fluid containing a nitric oxide-lowering agent in combination with the Na þ /K þ -ATPase inhibitor ouabain at a concentration selectively inhibiting a 2 /a 3 activity. Decline in a 2 /a 3 activity results in increased Ca 2þ uptake by internal stores of astrocytes, vascular myocytes, and pericytes since Ca 2þ outflux via plasmalemmal Na þ /Ca 2þ -exchanger declines. Augmented Ca 2þ mobilization from internal stores during spreading depolarization might enhance vasoconstriction, thus, contributing to spreading ischemia. Accordingly, spreading ischemia was significantly shortened when intracellular Ca 2þ stores were emptied by pre-treatment with thapsigargin, an inhibitor of the sarco(endo)plasmic reticulum Ca 2þ -ATPase (SERCA). These findings might have relevance for clinical conditions, in which spreading ischemia occurs such as delayed cerebral ischemia after subarachnoid hemorrhage.