Synaptic Zn2+ contributes to deleterious consequences of spreading depolarizations.

Spreading depolarizations (SDs) are profound waves of neuroglial depolarization that can propagate repetitively through injured brain. Recent clinical work has established SD as an important contributor to expansion of acute brain injuries and have begun to extend SD studies into other neurological disorders. A critical challenge is to determine how to selectively prevent deleterious consequences of SD. In the present study, we determined whether a wave of profound Zn2+ release is a key contributor to deleterious consequences of SD, and whether this can be targeted pharmacologically. Focal KCl microinjection was used to initiate SD in the CA1 region of the hippocampus in murine brain slices. An extracellular Zn2+ chelator with rapid kinetics (ZX1) increased SD propagation rates and improved recovery of extracellular DC potential shifts. Under conditions of metabolic compromise, tissues showed sustained impairment of functional and structural recovery following a single SD. ZX1 effectively improved recovery of synaptic potentials and intrinsic optical signals in these vulnerable conditions. Fluorescence imaging and genetic deletion of a presynaptic Zn2+ transporter confirmed synaptic release as the primary contributor to extracellular accumulation and deleterious consequences of Zn2+ during SD. These results demonstrate a role for synaptic Zn2+ release in deleterious consequences of SD and show that targeted extracellular chelation could be useful for disorders where repetitive SD enlarges infarcts in injured tissues.

Ketamine improves neuronal recovery following spreading depolarization in peri-infarct tissues.

Spreading depolarization (SD) has emerged as an important contributor to the enlargement of acute brain injuries. We previously showed that the N-methyl-D-aspartate receptor antagonist ketamine was able to prevent deleterious consequences of SD in brain slices, under conditions of metabolic compromise. The current study aimed to extend these observations into an in vivo stroke model, to test whether gradients of metabolic capacity lead to differential accumulation of calcium (Ca2+ ) following SD. In addition, we tested whether ketamine protects vulnerable tissuewhile allowing SD to propagate through surrounding undamaged tissue. Focal lesions were generated using a distal middle cerebral artery occlusion in mice, and clusters of SD were generated at 20 min intervals with remote microinjection of potassium chloride. SDs invading peri-infarct regions had significantly different consequences, depending on the distance from the infarct core. Proximal to the lesion, Ca2+ transients were extended, as compared with responses in better-perfused tissue more remote from the lesion. Extracellular potential shifts were also longer and hyperemia responses were reduced in proximal regions following SDs. Consistent with in vitro studies, ketamine, at concentrations that did not abolish the propagation of SD, reduced the accumulation of intracellular Ca2+ in proximal regions following an SD wave. These findings suggest that deleterious consequences of SD can be targeted in vivo, without requiring outright block of SD initiation and propagation.

Action Potentials Are Critical for the Propagation of Focally Elicited Spreading Depolarizations.

Spreading depolarizations (SDs) of gray matter occur in the brain in different pathologic conditions, and cause varying degrees of tissue damage depending on the extent of metabolic burden on the tissue. As might be expected for such large depolarizations, neurons exhibit bursts of action potentials (APs) as the wave propagates. However, the specific role of APs in SD propagation is unclear. This is potentially consequential, since sodium channel modulation has not been considered as a therapeutic target for SD-associated disorders, because of ambiguous experimental evidence. Using whole-cell electrophysiology and single-photon imaging in acute cortical slices from male C57Bl6 mice, we tested the effects of AP blockade on SDs generated by two widely used induction paradigms. We found that AP blockade using tetrodotoxin (TTX) restricted propagation of focally induced SDs, and significantly reduced the amplitude of neuronal depolarization, as well as its Ca2+ load. TTX also abolished the suppression of spontaneous synaptic activity that is a hallmark of focally induced SD. In contrast, TTX did not affect the propagation of SD induced by global superfusion of high [K+]e containing artificial CSF (ACSF). Thus, we show that voltage-gated sodium channel (Nav)-mediated neuronal AP bursts are critical for the propagation and downstream effects of focally induced SD but are less important when the ionic balance of the extracellular space is already compromised. In doing so we corroborate the notion that two different SD induction paradigms, each relevant to different clinical situations, vary significantly in their characteristics and potentially their response to treatment.SIGNIFICANCE STATEMENT Our findings suggest that voltage-gated sodium channel (Nav) channels have a critical role in the propagation and downstream neural effects of focally induced spreading depolarization (SD). As SDs are likely induced focally in many disease conditions, these studies support sodium channel modulation, a previously underappreciated therapeutic option in SD-associated disorders, as a viable approach.

Memantine Improves Recovery After Spreading Depolarization in Brain Slices and can be Considered for Future Clinical Trials.

BACKGROUND: Spreading depolarization (SD) has been identified as a key mediator of secondary lesion progression after acute brain injuries, and clinical studies are beginning to pharmacologically target SDs. Although initial work has focused on the N-Methyl-D-aspartate receptor antagonist ketamine, there is also interest in alternatives that may be better tolerated. We recently showed that ketamine can inhibit mechanisms linked to deleterious consequences of SD in brain slices. The present study tested the hypothesis that memantine improves recovery of brain slices after SD and explored the effects of memantine in a clinical case targeting SD. METHODS: For mechanistic studies, electrophysiological and optical recordings were made from hippocampal area CA1 in acutely prepared brain slices from mice. SDs were initiated by localized microinjection of K+ in conditions of either normal or reduced metabolic substrate availability. Memantine effects were assessed from intrinsic optical signals and extracellular potential recordings. For the clinical report, a subdural strip electrode was used for continuous electrocorticographic recording after the surgical evacuation of a chronic subdural hematoma. RESULTS: In brain slice studies, memantine (10-300 µM) did not prevent the initiation of SD, but impaired SD propagation rate and recovery from SD. Memantine reduced direct current (DC) shift duration and improved recovery of synaptic potentials after SD. In brain slices with reduced metabolic substrate availability, memantine reduced the evidence of structural disruption after the passage of SD. In our clinical case, memantine did not noticeably immediately suppress SD; however, it was associated with a significant reduction of SD duration and a reduction in the electrocorticographic (ECoG) suppression that occurs after SD. SD was completely suppressed, with improvement in neurological examination with the addition of a brief course of ketamine. CONCLUSIONS: These data extend recent work showing that N-Methyl-D-aspartate receptor antagonists can improve recovery from SD. These results suggest that memantine could be considered for future clinical trials targeting SD, and in some cases as an adjunct or alternative to ketamine.

Non-canonical glutamate signaling in a genetic model of migraine with aura.

Migraine with aura is a common but poorly understood sensory circuit disorder. Monogenic models allow an opportunity to investigate its mechanisms, including spreading depolarization (SD), the phenomenon underlying migraine aura. Using fluorescent glutamate imaging, we show that awake mice carrying a familial hemiplegic migraine type 2 (FHM2) mutation have slower clearance during sensory processing, as well as previously undescribed spontaneous "plumes" of glutamate. Glutamatergic plumes overlapped anatomically with a reduced density of GLT-1a-positive astrocyte processes and were mimicked in wild-type animals by inhibiting glutamate clearance. Plume pharmacology and plume-like neural Ca2+ events were consistent with action-potential-independent spontaneous glutamate release, suggesting plumes are a consequence of inefficient clearance following synaptic release. Importantly, a rise in basal glutamate and plume frequency predicted the onset of SD in both FHM2 and wild-type mice, providing a novel mechanism in migraine with aura and, by extension, the other neurological disorders where SD occurs.

Which Spreading Depolarizations Are Deleterious To Brain Tissue?

Spreading depolarizations (SDs) are profound disruptions of cellular homeostasis that slowly propagate through gray matter and present an extraordinary metabolic challenge to brain tissue. Recent work has shown that SDs occur commonly in human patients in the neurointensive care setting and have established a compelling case for their importance in the pathophysiology of acute brain injury. The International Conference on Spreading Depolarizations (iCSD) held in Boca Raton, Florida, in September of 2018 included a discussion session focused on the question of "Which SDs are deleterious to brain tissue?" iCSD is attended by investigators studying various animal species including invertebrates, in vivo and in vitro preparations, diseases of acute brain injury and migraine, computational modeling, and clinical brain injury, among other topics. The discussion included general agreement on many key issues, but also revealed divergent views on some topics that are relevant to the design of clinical interventions targeting SDs. A draft summary of viewpoints offered was then written by a multidisciplinary writing group of iCSD members, based on a transcript of the session. Feedback of all discussants was then formally collated, reviewed and incorporated into the final document. It is hoped that this report will stimulate collection of data that are needed to develop a more nuanced understanding of SD in different pathophysiological states, as the field continues to move toward effective clinical interventions.

Ketamine reduces deleterious consequences of spreading depolarizations.

Recent work has implicated spreading depolarization (SD) as a key contributor the progression of acute brain injuries, however development of interventions selectively targeting SD has lagged behind. Initial clinical intervention efforts have focused on observations that relatively high doses of the sedative agent ketamine can completely suppress SD. However, blocking propagation of SD could theoretically prevent beneficial effects of SD in surrounding brain regions. Selective targeting of deleterious consequences of SD (rather than abolition) could be a useful adjunct approach, and be achieved with lower ketamine concentrations. We utilized a brain slice model to test whether deleterious consequences of SD could be prevented by ketamine, using concentrations that did not prevent the initiation and propagation of SD. Studies were conducted using murine brain slices, with focal KCl as an SD stimulus. Consequences of SD were assessed with electrophysiological and imaging measures of ionic and synaptic recovery. Under control conditions, ketamine (up to 30 µM) did not prevent SD, but significantly reduced neuronal Ca2+ loading and the duration of associated extracellular potential shifts. Recovery of postsynaptic potentials after SD was also significantly accelerated. When SD was evoked on a background of mild metabolic compromise, neuronal recovery was substantially impaired. Under compromised conditions, the same concentrations of ketamine reduced ionic and metabolic loading during SD, sufficient to preserve functional recovery after repetitive SDs. These results suggest that lower concentrations of ketamine could be utilized to prevent damaging consequences of SD, while not blocking them outright and thereby preserving potentially protective effects of SD.

Sulfate-reducing bacteria impairs working memory in mice.

The ability of gut microbes to bi-directionally communicate with the brain and vice versa form the basis of the gut microbiome-central nervous system axis. It has been shown that inoculation with pathogenic gut bacteria alters the behavior of mice; however, it is not known whether or not non-pathogenic resident microbes have similar effects. In this study, we tested the hypothesis that the administration of sulfate-reducing bacteria (SRB), a specific group of resident gut bacteria that generate hydrogen sulfide (H2S), impair learning and memory performance in mice tested in an 8-arm radial maze and Morris water maze. We found that mice spent more time in the center of the maze when they were gavaged with live SRB as compared to mice given saline (control), lactulose+mannitol (L/M), or killed SRB. SRB-gavaged mice were also tested using the Morris water maze and were found to take longer to complete the test, spend more time further from the platform, and have a longer path length to reach the platform. This effect of SRB on maze performance was associated with a higher concentration of H2S in the small intestine and cecum. We conclude that SRB, a specific resident gut bacterial species, could impair cognitive function in mice.