R. David Andrew

Spreading depolarizations (SDs) are waves of mass depolarization that propagate through gray matter following Na ⁺ /K ⁺ -ATPase (NKA) failure because of stroke, traumatic brain injury or sudden cardiac arrest. SDs expand the initial site of neuronal injury and worsen clinical outcomes. The molecular events underlying SD initiation and propagation are not well understood. In this rodent study, we hypothesized that gray matter stressed by oxygen/glucose deprivation (OGD) releases a compound(s) that promotes SD, which we term a spreading depolarization activator (SD a ). We used rat brain slices incubated in artificial cerebrospinal fluid (aCSF) and subjected to OGD to release a putative SDa. The aCSF was collected either prior to (“Pre-SD aCSF”) or 10 min after initiation of OGD conditions (“Post-SD _OGD aCSF”). These solutions were then separately superfused over a healthy, naïve (non-stressed) brain slice. Post-SD _OGD aCSF (with re-normalized O ₂ and glucose) evoked SD in 82.35% of the naïve brain slices ( n = 17) whereas Pre-SD aCSF evoked no SD in 10 naïve slices. Then to investigate the NKA as a potential target of the SD a , we used a hemolysis assay, comparing the effects of Pre- or Post-SD _OGD aCSF on red blood cell (RBC) lysis and compared it to the known hemolytic effect of the NKA-specific inhibitor, palytoxin. Post-SD _OGD aCSF evoked neither swelling nor lysis of RBCs on its own. However, when a sub-threshold concentration (0.01–0.02 nM) of the specific NKA inhibitor palytoxin (PLTX) was added, a striking “priming” effect was observed, whereby Post-SD _OGD aCSF evoked a highly significant increase in both RBC swelling and then hemolysis, compared to Pre-SD aCSF. High pressure liquid chromatography (HPLC) experiments show a several-fold increase in released molecules post-SD vs pre-SD. Overall, this study provides support for SD a release capable of inducing SD-associated swelling in brain slices and, when combined with a trace amount of PLTX, swelling/hemolysis of RBCs caused by NKA inhibition. A greater understanding of the molecular events underlying SD should identify novel targets to reduce recurrent SD-evoked neuronal injury under ischemic conditions.

With stroke, traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug to aid brain recovery. Within 2 min of severe ischemia, a wave of spreading depolarization (SD) propagates through affected gray matter. More SDs arise over hours, expanding the injury. This period represents a therapeutic window to inhibit recurring SD and reduce neuronal damage, but we do not understand the underlying molecular sequence. Here, we argue for a novel molecule to target.

Spreading depolarization (SD) is a critical mechanism underlying central nervous system (CNS) shutdown and injury under stress, yet the initiating ion channel remains unknown. Here, we used the marine poison palytoxin (PLTX), which converts the sodium-potassium ATPase (NKA) into an open channel, to initiate SD in intact locust CNS. We show for the first time that PLTX-induced SD is rapid and recoverable in vivo, providing support that NKA conversion to a channel may be the SD-initiating mechanism.

The gray matter of the higher brain undergoes spreading depolarization (SD) in response to the increased metabolic demand of ischemia, promoting acute neuronal injury and death. The mechanism linking ischemic failure of the Na ⁺ /K ⁺ ATPase (NKA) to the subsequent onset of a large inward current driving SD in neurons has remained a mystery because blockade of conventional channels does not prevent SD nor ischemic death. The marine poison palytoxin (PLTX) specifically binds the NKA transporter at extremely low concentrations, converting it to an open cationic channel, causing sudden neuronal Na ⁺ influx and K ⁺ efflux. Pump failure and induction of a strong inward current should induce dramatic SD-like activity. Indeed,1-10 nM PLTX applied to live coronal brain slices induces a propagating depolarization remarkably like SD induced by oxygen/glucose deprivation (OGD) as revealed by imaging. This PLTX depolarization (PD) mimicked other effects of OGD. In neocortex, as the elevated LT front passed by an extracellular pipette, a distinct negative DC shift was recorded, indicating cell depolarization, whether induced by OGD or by bath PLTX. Either treatment induced strong SD-like responses in the same higher and lower brain regions. Further, we imaged identical real-time OGD-SD or PD effects upon live pyramidal neurons using 2-photon microscopy. Taken together, these findings support our proposal that, like most biological poisons, PLTX mimics (and takes advantage of) a biological process,ie is brain ischemia. An endogenous PLTX-like molecule may open the NKA to evoke Na ⁺ influx/K ⁺ efflux that drive SD and the ensuing neuronal damage in its wake. New and Noteworthy: With stroke, traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug to aid brain protection and recovery. Within 2 minutes of severe ischemia, a wave of spreading depolarization (SD) propagates through gray matter. More SDs arise over hours, expanding injury. This period represents a therapeutic window to inhibit recurring SD and reduce damage but we do not understand the molecular sequence. Here we argue for a novel molecule to target.

Background: When a patient arrives in the emergency department following a stroke, a traumatic brain injury, or sudden cardiac arrest, there is no therapeutic drug available to help protect their jeopardized neurons. One crucial reason is that we have not identified the molecular mechanisms leading to electrical failure, neuronal swelling, and blood vessel constriction in newly injured gray matter. All three result from a process termed spreading depolarization (SD). Because we only partially understand SD, we lack molecular targets and biomarkers to help neurons survive after losing their blood flow and then undergoing recurrent SD.Methods: In this review, we introduce SD as a single or recurring event, generated in gray matter following lost blood flow, which compromises the Na⁺/K⁺pump. Electrical recovery from each SD event requires so much energy that neurons often die over minutes and hours following initial injury, independent of extracellular glutamate.Results: We discuss how SD has been investigated with various pitfalls in numerous experimental preparations, how overtaxing the Na⁺/K⁺ATPase elicits SD. Elevated K⁺or glutamate are unlikely natural activators of SD. We then turn to the properties of SD itself, focusing on its initiation and propagation as well as on computer modeling.Conclusions: Finally, we summarize points of consensus and contention among the authors as well as where SD research may be heading. In an accompanying review, we critique the role of the glutamate excitotoxicity theory, how it has shaped SD research, and its questionable importance to the study of early brain injury as compared with SD theory.

Background: Within 2 min of severe ischemia, spreading depolarization (SD) propagates like a wave through compromised gray matter of the higher brain. More SDs arise over hours in adjacent tissue, expanding the neuronal damage. This period represents a therapeutic window to inhibit SD and so reduce impending tissue injury. Yet most neuroscientists assume that the course of early brain injury can be explained by glutamate excitotoxicity, the concept that immediate glutamate release promotes early and downstream brain injury. There are many problems with glutamate release being the unseen culprit, the most practical being that the concept has yielded zero therapeutics over the past 30 years. But the basic science is also flawed, arising from dubious foundational observations beginning in the 1950s Methods: Literature pertaining to excitotoxicity and to SD over the past 60 years is critiqued. Results: Excitotoxicity theory centers on the immediate and excessive release of glutamate with resulting neuronal hyperexcitation. This instigates poststroke cascades with subsequent secondary neuronal injury. By contrast, SD theory argues that although SD evokes some brief glutamate release, acute neuronal damage and the subsequent cascade of injury to neurons are elicited by the metabolic stress of SD, not by excessive glutamate release. The challenge we present here is to find new clinical targets based on more informed basic science. This is motivated by the continuing failure by neuroscientists and by industry to develop drugs that can reduce brain injury following ischemic stroke, traumatic brain injury, or sudden cardiac arrest. One important step is to recognize that SD plays a central role in promoting early neuronal damage. We argue that uncovering the molecular biology of SD initiation and propagation is essential because ischemic neurons are usually not acutely injured unless SD propagates through them. The role of glutamate excitotoxicity theory and how it has shaped SD research is then addressed, followed by a critique of its fading relevance to the study of brain injury. Conclusions: Spreading depolarizations better account for the acute neuronal injury arising from brain ischemia than does the early and excessive release of glutamate.

Background: Neuronal damage resulting from increased oxidative stress is important in the development of late onset/age-related Alzheimer’s disease (LOAD). We have developed an oxidative stress–related mouse model of LOAD based on gene deletion of aldehyde dehydrogenase 2 (ALDH2), an enzyme important for the detoxification of endogenous aldehydes arising from lipid peroxidation. Compared to wildtype (WT) mice, the knockout (KO) mice exhibit AD-like pathologies and a progressive decline in recognition and spatial memory. This progression presumably has a morphological basis induced by oxidative damage. Objective: We performed morphometric analyses in the dorsal hippocampal CA1 region (dCA1) to determine if altered neuronal structure can help account for the progressive cognitive impairment in 3- to 12-month-old KO mice. Methods: Dendritic morphology was quantitatively analyzed by branched structured analysis and Sholl analysis following Golgi-Cox staining in WT mice (148 neurons) versus KO mice (180 neurons). Results: The morphology and complexity of dCA1 pyramidal neurons were similar at age 3 months in WTs and KOs. However, by 6 months there were significant reductions in apical and basal dendritic length, dendrite complexity, and spine density in KO versus WT mice that were maintained through ages 9 and 12 months. Immunostaining for protein adducts of the lipid peroxidation product 4-hydroxynonenal revealed significant increases in staining in dCA1 (but not ventral CA1) by 3 months, increasing through 12 months. Conclusion: This specific and progressive increase in dCA1 oxidative damage preceded detectable synaptic trimming in KO mice, in keeping with studies showing that lesions to dorsal hippocampus primarily impair cognitive memory.

The study of late-onset (sporadic) Alzheimer’s disease (LOAD) has lacked animal models where impairments develop with aging. Oxidative stress promotes LOAD, so we have developed an oxidative stress-based model of age-related cognitive impairment based on gene deletion of aldehyde dehydrogenase 2 (ALDH2). This enzyme is important for the detoxification of endogenous aldehydes arising from lipid peroxidation. Compared to wildtype (WT) mice, the knockout (KO) mice exhibit a progressive decline in recognition and spatial memory and AD-like pathologies. Here we performed morphometric analyses in the dorsal and ventral hippocampal CA1 regions (dCA1 and vCA1) as well as in overlying primary sensory cortex to determine if altered neuronal structure can help account for the cognitive impairment in 12-month old KO mice. Dendritic morphology was quantitatively analyzed following Golgi-Cox staining using 9 WT mice (108 neurons) and 15 KO mice (180 neurons). Four pyramidal neurons were traced per mouse in each region, followed by branched structured analysis and Sholl analysis. Compared to WT controls, the morphology and complexity of dCA1 pyramidal neurons from KOs showed significant reductions in apical and basal dendritic length, dendrite intersections, ends, and nodes. As well, spine density along dorsal CA1 apical dendrites was significantly lower in KO versus WT. In contrast, pyramidal arborization in the vCA1 and primary sensory cortex were only minimally reduced in KO versus WT mice. These data suggest a region-specific vulnerability to oxidative stress-induced damage and/or a major and specific reduction in synaptic input to the pyramidal neurons of the dorsal hippocampus. This is in keeping with studies showing that lesions to the dorsal hippocampus impair primarily cognitive memory whereas ventral hippocampal lesions cause deficits in stress, emotion, and affect.

Whereas many studies have examined the properties of the compromised neocortex in the first several days following ischemia, there is less information regarding the initial 12 h poststroke. In this study we examined live mouse neocortical slices harvested immediately and 12 h after a 30-min middle cerebral artery occlusion (MCAo). We compared nonischemic and ischemic hemispheres with regard to the propensity for tissue swelling and for generating spreading depolarization (SD), as well as evoked synaptic responses and single pyramidal neuron electrophysiological properties. We observed spontaneous SD in 7% of slices on the nonstroked side and 25% in the stroked side following the 30-min MCAo. Spontaneous SD was rare in 12-h recovery slices. The region of the ischemic core and surround in slices was not susceptible to SD induced by oxygen and glucose deprivation. At the neuronal level, neocortical gray matter is surprisingly unaltered in brain slices harvested immediately poststroke. However, by 12 h, the fields of pyramidal and striatal neurons that comprise the infarcted core are electrophysiologically silent because the majority are morphologically devastated. Yet, there remains a subset of diffusely distributed “healthy” pyramidal neurons in the core at 12 h post-MCAo that persist for days poststroke. Their intact electrophysiology and dendritic morphology indicate a surprisingly selective resilience to stroke at the neuronal level. NEW & NOTEWORTHY It is generally accepted that the injured core region of the brain resulting from a focal stroke contains no functioning neurons. Our study shows that some neurons, although surrounded by devastated neighbors, can maintain their structure and electrical activity. This surprising finding raises the possibility of discovering how these neurons are protected to pinpoint new strategies for reducing stroke injury.