Vagus nerve stimulation reduces spreading depolarization burden and cortical infarct volume in a rat model of stroke.

Cortical spreading depolarization (SD) waves negatively affect neuronal survival and outcome after ischemic stroke. We here aimed to investigate the effects of vagus nerve stimulation (VNS) on SDs in a rat model of focal ischemia. To this end, we delivered non-invasive VNS (nVNS) or invasive VNS (iVNS) during permanent middle cerebral artery occlusion (MCAO), and found that both interventions significantly reduced the frequency of SDs in the cortical peri-infarct area compared to sham VNS, without affecting relative blood flow changes, blood pressure, heart rate or breathing rate. In separate groups of rats subjected to transient MCAO, we found that cortical stroke volume was reduced 72 h after transient MCAO, whereas stroke volume in the basal ganglia remained unchanged. In rats treated with nVNS, motor outcome was improved 2 days after transient MCAO, but was similar to sham VNS animals 3 days after ischemia. We postulate that VNS may be a safe and efficient intervention to reduce the clinical burden of SD waves in stroke and other conditions.

Local Resting Ca2+ Controls the Scale of Astroglial Ca2+ Signals.

Astroglia regulate neurovascular coupling while engaging in signal exchange with neurons. The underlying cellular machinery is thought to rely on astrocytic Ca2+ signals, but what controls their amplitude and waveform is poorly understood. Here, we employ time-resolved two-photon excitation fluorescence imaging in acute hippocampal slices and in cortex in vivo to find that resting [Ca2+] predicts the scale (amplitude) and the maximum (peak) of astroglial Ca2+ elevations. We bidirectionally manipulate resting [Ca2+] by uncaging intracellular Ca2+ or Ca2+ buffers and use ratiometric imaging of a genetically encoded Ca2+ indicator to establish that alterations in resting [Ca2+] change co-directionally the peak level and anti-directionally the amplitude of local Ca2+ transients. This relationship holds for spontaneous and for induced (for instance by locomotion) Ca2+ signals. Our findings uncover a basic generic rule of Ca2+ signal formation in astrocytes, thus also associating the resting Ca2+ level with the physiological "excitability" state of astroglia.

Stroke target identification guided by astrocyte transcriptome analysis.

Astrocytes support normal brain function, but may also contribute to neurodegeneration when they become reactive under pathological conditions such as stroke. However, the molecular underpinnings of this context-dependent interplay between beneficial and detrimental properties in reactive astrogliosis have remained incompletely understood. Therefore, using the RiboTag technique, we immunopurified translating mRNAs specifically from astrocytes 72 hr after transient middle cerebral artery occlusion in mice (tMCAO), thereby generating a stroke-specific astroglial translatome database. We found that compared to control brains, reactive astrocytes after tMCAO show an enrichment of transcripts linked to the A2 phenotype, which has been associated with neuroprotection. However, we found that astrocytes also upregulate a large number of potentially neurotoxic genes. In total, we identified the differential expression of 1,003 genes and 38 transcription factors, of which Stat3, Sp1, and Spi1 were the most prominent. To further explore the effects of Stat3-mediated pathways on stroke pathogenesis, we subjected mice with an astrocyte-specific conditional deletion of Stat3 to tMCAO, and found that these mice have reduced stroke volume and improved motor outcome 72 hr after focal ischemia. Taken together, our study extends the emerging database of novel astrocyte-specific targets for stroke therapy, and supports the role of astrocytes as critical safeguards of brain function in health and disease.

Reverse NCX Attenuates Cellular Sodium Loading in Metabolically Compromised Cortex.

In core regions of ischemic stroke, disruption of blood flow causes breakdown of ionic gradients and, ultimately, calcium overload and cell death. In the surrounding penumbra, cells may recover upon reperfusion, but recovery is hampered by additional metabolic demands imposed by peri-infarct depolarizations (PIDs). There is evidence that sodium influx drives PIDs, but no data exist on PID-related sodium accumulations in vivo. Here, we found that PIDs in mouse neocortex are associated with propagating sodium elevations in neurons and astrocytes. Similar transient sodium elevations were induced in acute tissue slices by brief chemical ischemia. Blocking NMDA-receptors dampened sodium and accompanying calcium loads of neurons in tissue slices, while inhibiting glutamate transport diminished sodium influx into astrocytes, but amplified neuronal sodium loads. In both cell types, inhibition of sodium/calcium exchange (NCX) increased sodium transients. Blocking NCX also significantly reduced calcium transients, a result confirmed in vivo. Our study provides the first quantitative data on sodium elevations in peri-infarct regions in vivo. They suggest that sodium influx drives reversal of NCX, triggering a massive secondary calcium elevation while promoting export of sodium. Reported neuroprotective effects of NCX activity in stroke models might thus be related to its dampening of ischemia-induced sodium loading.

TRPV4 channels contribute to calcium transients in astrocytes and neurons during peri-infarct depolarizations in a stroke model.

Stroke is one of the leading causes of death and long-term disability. In the penumbra, that is, the area surrounding the infarct core, peri-infarct depolarizations (PIDs) are accompanied by strong intracellular calcium elevations in astrocytes and neurons, thereby negatively affecting infarct size and clinical outcome. The dynamics of PIDs and the cellular pathways that are involved during PID formation and progression remain incompletely understood. We have previously shown that inositol triphosphate-gated calcium release from internal stores is a major component of PID-related astroglial calcium signals, but whether external calcium influx through membrane-localized channels also contributes to PIDs has remained unclear. In this study, we investigated the role of two astroglial membrane channels, transient receptor vanilloid 4 (TRPV4) channel and aquaporin-4 (AQP4). We combined in vivo multiphoton microscopy, electrophysiology as well as laser speckle contrast imaging with the middle cerebral artery occlusion stroke model. Using knockout mice and pharmacological inhibitors, we found that TRPV4 channels contribute to calcium influx into astrocytes and neurons and subsequent extracellular glutamate accumulation during PIDs. AQP4 neither influenced PID-related calcium signals nor PID-related edema of astrocyte somata. Both channels did not alter the dynamics, frequency and cerebrovascular response of PIDs in the penumbra. These data indicate that TRPV4 channels may represent a potential target to ameliorate the PID-induced calcium overload of astrocytes and neurons during acute stroke.

Differential effects of energy deprivation on intracellular sodium homeostasis in neurons and astrocytes.

The maintenance of a low intracellular sodium concentration by the Na+ /K+ -ATPase (NKA) is critical for brain function. In both neurons and glial cells, NKA activity is required to counteract changes in the sodium gradient due to opening of voltage- and ligand-gated channels and/or activation of sodium-dependent secondary active transporters. Because NKA consumes about 50% of cellular ATP, sodium homeostasis is strictly dependent on an intact cellular energy metabolism. Despite the high energetic costs of electrical signaling, neurons do not contain significant energy stores themselves, but rely on a close metabolic interaction with surrounding astrocytes. A disruption of energy supply as observed during focal ischemia causes a rapid drop in ATP in both neurons and astrocytes. There is accumulating evidence that dysregulation of intracellular sodium is an inherent consequence of a reduction in cellular ATP, triggering secondary failure of extra- and intracellular homeostasis of other ions -in particular potassium, calcium, and protons- and thereby promoting excitotoxicity. The characteristics, cellular mechanisms and direct consequences of harmful sodium influx, however, differ between neurons and astrocytes. Moreover, recent work has shown that an intact astrocyte metabolism and sodium homeostasis are critical to maintain the sodium homeostasis of surrounding neurons as well as their capacity to recover from imposed sodium influx. Understanding the mechanisms of sodium increases upon metabolic failure and the differential responses of neurons and glial cells as well as their metabolic interactions will be critical to fully unravel the events causing cellular malfunction, failure and cell death following energy depletion. © 2017 Wiley Periodicals, Inc.

Astrocytic calcium release mediates peri-infarct depolarizations in a rodent stroke model.

Stroke is one of the most common diseases and a leading cause of death and disability. Cessation of cerebral blood flow (CBF) leads to cell death in the infarct core, but tissue surrounding the core has the potential to recover if local reductions in CBF are restored. In these areas, detrimental peri-infarct depolarizations (PIDs) contribute to secondary infarct growth and negatively affect stroke outcome. However, the cellular pathways underlying PIDs have remained unclear. Here, we have used in vivo multiphoton microscopy, laser speckle imaging of CBF, and electrophysiological recordings in a mouse model of focal ischemia to demonstrate that PIDs are associated with a strong increase of intracellular calcium in astrocytes and neurons. We found that astroglial calcium elevations during PIDs are mediated by inositol triphosphate receptor type 2-dependent (IP3R2-dependent) release from internal stores. Importantly, Ip3r2-deficient mice displayed a reduction of PID frequency and overall PID burden and showed increased neuronal survival after stroke. These effects were not related to local CBF changes in response to PIDs. However, we showed that the release and extracellular accumulation of glutamate during PIDs is strongly curtailed in Ip3r2-deficient mice, resulting in ameliorated calcium overload in neurons and astrocytes. Together, these data implicate astroglial calcium pathways as potential targets for stroke therapy.

Astroglial NF-kB contributes to white matter damage and cognitive impairment in a mouse model of vascular dementia.

Vascular cognitive impairment is the second most common form of dementia. The pathogenic pathways leading to vascular cognitive impairment remain unclear but clinical and experimental data have shown that chronic reactive astrogliosis occurs within white matter lesions, indicating that a sustained pro-inflammatory environment affecting the white matter may contribute towards disease progression. To model vascular cognitive impairment, we induced prolonged mild cerebral hypoperfusion in mice by bilateral common carotid artery stenosis. This chronic hypoperfusion resulted in reactive gliosis of astrocytes and microglia within white matter tracts, demyelination and axonal degeneration, consecutive spatial memory deficits, and loss of white matter integrity, as measured by ultra high-field magnetic resonance diffusion tensor imaging. White matter astrogliosis was accompanied by activation of the pro-inflammatory transcription factor nuclear factor (NF)-kB in reactive astrocytes. Using mice expressing a dominant negative inhibitor of NF-kB under the control of the astrocyte-specific glial fibrillary acid protein (GFAP) promoter (GFAP-IkBα-dn), we found that transgenic inhibition of astroglial NF-kB signaling ameliorated gliosis and axonal loss, maintained white matter structural integrity, and preserved memory function. Collectively, our results imply that pro-inflammatory changes in white matter astrocytes may represent an important detrimental component in the pathogenesis of vascular cognitive impairment, and that targeting these pathways may lead to novel therapeutic strategies.