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During brain ischemia, excitotoxicity and peri-infarct depolarization injuries occur and cause cerebral tissue damage. Indeed, anoxic depolarization, consisting of massive neuronal depolarization due to the loss of membrane ion gradients, occurs in vivo or in vitro during an energy failure. The neuromodulator adenosine is released in huge amounts during cerebral ischemia and exerts its effects by activating specific metabotropic receptors, namely: A1, A2A, A2B, and A3. The A2A receptor subtype is highly expressed in striatal medium spiny neurons, which are particularly susceptible to ischemic damage. Evidence indicates that the A2A receptors are upregulated in the rat striatum after stroke and the selective antagonist SCH58261 protects from exaggerated glutamate release within the first 4 hours from the insult and alleviates neurological impairment and histological injury in the following 24 hours. We recently added new knowledge to the mechanisms by which the adenosine A2A receptor subtype participates in ischemia-induced neuronal death by performing patch-clamp recordings from medium spiny neurons in rat striatal brain slices exposed to oxygen and glucose deprivation. We demonstrated that the selective block of A2A receptors by SCH58261 significantly reduced ionic imbalance and delayed the anoxic depolarization in medium spiny neurons during oxygen and glucose deprivation and that the mechanism involves voltage-gated K+ channel modulation and a presynaptic inhibition of glutamate release by the A2A receptor antagonist. The present review summarizes the latest findings in the literature about the possibility of developing selective ligands of A2A receptors as advantageous therapeutic tools that may contribute to counteracting neurodegeneration after brain ischemia.

In recent years, the use of multi-target compounds has become an increasingly pursued strategy to treat complex pathologies, including cerebral ischemia. Adenosine and its receptors (A1AR, A2AAR, A2BAR, A3AR) are known to play a crucial role in synaptic transmission either in normoxic or ischemic-like conditions. Previous data demonstrate that the selective antagonism of A2AAR or A2BAR delays anoxic depolarization (AD) appearance, an unequivocal sign of neuronal injury induced by a severe oxygen-glucose deprivation (OGD) insult in the hippocampus. Furthermore, the stimulation of A2AARs or A2BARs by respective selective agonists, CGS21680 and BAY60-6583, increases pre-synaptic neurotransmitter release, as shown by the decrease in paired-pulse facilitation (PPF) at Schaffer collateral-CA1 synapses. In the present research, we investigated the effect/s of the newly synthesized dual A2AAR/A2BAR antagonist, P626, in preventing A2AAR- and/or A2BAR-mediated effects by extracellular recordings of synaptic potentials in the CA1 rat hippocampal slices. We demonstrated that P626 prevented PPF reduction induced by CGS21680 or BAY60-6583 and delayed, in a concentration-dependent manner, AD appearance during a severe OGD. In conclusion, P626 may represent a putative neuroprotective compound for stroke treatment with the possible translational advantage of reducing side effects and bypassing differences in pharmacokinetics due to combined treatment. [ABSTRACT FROM AUTHOR]

Background and Purpose: Ischaemia is known to cause massive neuronal depolarization, termed anoxic depolarization (AD), due to energy failure and loss of membrane ion gradients. The neuromodulator adenosine accumulates extracellularly during ischaemia and activates four metabotropic receptors: A1 , A2A , A2B and A3 . Striatal medium spiny neurons (MSNs) express high levels of A2A receptors and are particularly vulnerable to ischaemic insults. A2A Receptor blockade reduces acute striatal post-ischaemic damage but the cellular mechanisms involved are still unknown.Experimental Approach: We performed patch-clamp recordings of MSNs in rat striatal slices subjected to oxygen and glucose deprivation (OGD) to investigate the effects of A2A receptor ligands or ion channel blockers on AD and OGD-induced ionic imbalance, measured as a positive shift in Erev of ramp currents.Key Results: Our data indicate that the A2A receptor antagonist SCH58261 (10 μM) significantly attenuated ionic imbalance and AD appearance in MSNs exposed to OGD. The K+ channel blocker Ba2+ (2 mM) or the Na+ channel blocker tetrodotoxin (1 μM) exacerbated and attenuated, respectively, OGD-induced changes. Spontaneous excitatory post-synaptic current (sEPSC) analysis in MSNs revealed that the A2A receptor agonist CGS21680 (1 μM) prevented OGD-induced decrease of sEPSCs within the first 5 min of the insult, an effect shared by the K+ channel blocker Ba2+ , indicating facilitated glutamate release.Conclusion and Implications: Adenosine, released during striatal OGD, activates A2A receptors that may exacerbate OGD-induced damage through K+ channel inhibition. Our results could help to develop A2A receptor-selective therapeutic tools for the treatment of brain ischaemia. [ABSTRACT FROM AUTHOR]

UnlabelledSpreading depolarizations (SDs) are recognized as actors in neurological disorders as diverse as migraine and traumatic brain injury (TBI). Migraine aura involves sensory percepts, suggesting that sensory cortices might be intrinsically susceptible to SDs. We used optical imaging, MRI, and field potential and potassium electrode recordings in mice and electrocorticographic recordings in humans to determine the susceptibility of different brain regions to SDs. Optical imaging experiments in mice under isoflurane anesthesia showed that both cortical spreading depression and terminal anoxic depolarization arose preferentially in the whisker barrel region of parietal sensory cortex. MRI recordings under isoflurane, ketamine/xylazine, ketamine/isoflurane, and urethane anesthesia demonstrated that the depolarizations did not propagate from a subcortical source. Potassium concentrations showed larger increases in sensory cortex, suggesting a mechanism of susceptibility. Sensory stimulation biased the timing but not the location of depolarization onset. In humans with TBI, there was a trend toward increased incidence of SDs in parietal/temporal sensory cortex compared with other regions. In conclusion, SDs are inducible preferentially in primary sensory cortex in mice and most likely in humans. This tropism can explain the predominant sensory phenomenology of migraine aura. It also demonstrates that sensory cortices are vulnerable in brain injury.Significance statementSpreading depolarizations (SDs) are involved in neurologic disorders as diverse as migraine and traumatic brain injury. In migraine, the nature of aura symptoms suggests that sensory cortex may be preferentially susceptible. In brain injury, SDs occur at a vulnerable time, during which the issue of sensory stimulation is much debated. We show, in mouse and human, that sensory cortex is more susceptible to SDs. We find that sensory stimulation biases the timing but not the location of the depolarizations. Finally, we show a relative impairment of potassium clearance in sensory cortex, providing a potential mechanism for the susceptibility. Our data help to explain the sensory nature of the migraine aura and reveal that sensory cortices are vulnerable in brain injury.

Ligands of the Gi protein-coupled adenosine A3 receptor (A3R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A3R. The present review summarizes information on the effect of latest-generation A3R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A3R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed. [ABSTRACT FROM AUTHOR]

Systematic administration of anti-inflammatory cytokine interleukin 4 (IL-4) has been shown to improve recovery after cerebral ischemic stroke. However, whether IL-4 affects neuronal excitability and how IL-4 improves ischemic injury remain largely unknown. Here we report the neuroprotective role of endogenous IL-4 in focal cerebral ischemia–reperfusion (I/R) injury. In multi-electrode array (MEA) recordings, IL-4 reduces spontaneous firings and network activities of mouse primary cortical neurons. IL-4 mRNA and protein expressions are upregulated after I/R injury. Genetic deletion of Il-4 gene aggravates I/R injury in vivo and exacerbates oxygen-glucose deprivation (OGD) injury in cortical neurons. Conversely, supplemental IL-4 protects Il-4−/− cortical neurons against OGD injury. Mechanistically, cortical pyramidal and stellate neurons common for ischemic penumbra after I/R injury exhibit intrinsic hyperexcitability and enhanced excitatory synaptic transmissions in Il-4−/− mice. Furthermore, upregulation of Nav1.1 channel, and downregulations of KCa3.1 channel and α6 subunit of GABAA receptors are detected in the cortical tissues and primary cortical neurons from Il-4−/− mice. Taken together, our findings demonstrate that IL-4 deficiency results in neural hyperexcitability and aggravates I/R injury, thus activation of IL-4 signaling may protect the brain against the development of permanent damage and help recover from ischemic injury after stroke.

• Time between anoxic depolarization (AD) and re-oxygenation dictates degree of electrophysiological recovery after anoxia. • Adenosine A 1 receptor antagonism has no effect on AD latency or the potential for recovery. • Antagonism of AMPA- and NMDA-receptors prolongs AD latency and flattens AD waves. • AD latency is longer in CA1 when disconnected from CA3. • AD latency in CA1 is protracted in female mice compared to male mice. Ischemic stroke remains the third leading cause of death and leading cause of adult disability worldwide. A key event in the pathophysiology of stroke is the anoxic depolarization (AD) of neurons in the ischemic core. Previous studies have established that both the latency to AD and the time spent in AD prior to re-oxygenation are predictors of neuronal death. The present studies used hippocampal slices from male and female mice to investigate the electrophysiological events that affect latency to AD after oxygen deprivation. The results confirm that the epoch between AD and re-oxygenation largely determines the magnitude of synaptic recovery after anoxic challenge. Using a selective antagonist of adenosine A 1 receptors, we also confirmed that adenosine released during anoxia (ANOX) suppresses synaptic glutamate release; however, this action has no effect on AD latency or the potential for post-anoxic recovery of synaptic transmission. In contrast, antagonism of AMPA- and NMDA-type glutamate receptors significantly prolongs the latency to AD and alters the speed and synchrony of associated depolarizing waves. Experiments using slices with fields Cornu ammonis 3 (CA3) and Cornu ammonis 1 (CA1) disconnected showed that AD latency is longer in CA1 than in CA3; however, the early AD in CA3 is propagated to CA1 in intact slices. Finally, AD latency in CA1 was found to be longer in slices from female mice than in those from age-matched male mice. The results have implications for stroke prevention and for understanding brain adaptations in hypoxia-tolerant animals. [ABSTRACT FROM AUTHOR]

Systematic administration of anti-inflammatory cytokine interleukin 4 (IL-4) has been shown to improve recovery after cerebral ischemic stroke. However, whether IL-4 affects neuronal excitability and how IL-4 improves ischemic injury remain largely unknown. Here we report the neuroprotective role of endogenous IL-4 in focal cerebral ischemia–reperfusion (I/R) injury. In multi-electrode array (MEA) recordings, IL-4 reduces spontaneous firings and network activities of mouse primary cortical neurons. IL-4 mRNA and protein expressions are upregulated after I/R injury. Genetic deletion of Il-4 gene aggravates I/R injury in vivo and exacerbates oxygen-glucose deprivation (OGD) injury in cortical neurons. Conversely, supplemental IL-4 protects Il-4 −/− cortical neurons against OGD injury. Mechanistically, cortical pyramidal and stellate neurons common for ischemic penumbra after I/R injury exhibit intrinsic hyperexcitability and enhanced excitatory synaptic transmissions in Il-4 −/− mice. Furthermore, upregulation of Nav1.1 channel, and downregulations of KCa3.1 channel and α 6 subunit of GABA A receptors are detected in the cortical tissues and primary cortical neurons from Il-4 −/− mice. Taken together, our findings demonstrate that IL-4 deficiency results in neural hyperexcitability and aggravates I/R injury, thus activation of IL-4 signaling may protect the brain against the development of permanent damage and help recover from ischemic injury after stroke. This study reveals a previously unknown mechanism by which IL-4 deficiency causes neural hyperexcitability and enhances neuronal excitatory transmissions. Supplementing IL-4 might be beneficial for improvement of functional recovery after brain ischemia injury. Image 1 [ABSTRACT FROM AUTHOR]

Soon after exposure to hypoxia or ischemia, neurons in cortical tissues undergo massive anoxic depolarization (AD). This precipitous event is preceded by more subtle neuronal changes, including enhanced excitatory and inhibitory synaptic transmitter release. Here, we have used patch-in-slice techniques to identify the earliest effects of acute hypoxia on the synaptic and intrinsic properties of Layer 5 neurons, to determine their time course and to evaluate the role of glutamate receptors in their generation. Coronal slices of mouse somatosensory cortex were maintained at 36 °C in an interface chamber and challenged with episodes of hypoxia. In recordings with cell-attached electrodes, the open probability of Ca2+-dependent BK channels began to increase within seconds of hypoxia onset, indicating a sharp rise in [Ca2+]i just beneath the membrane. By using a high concentration of K+ in the pipette, we simultaneously monitored the membrane potential and showed that the [Ca2+]i rise was not associated with membrane depolarization. The earliest hypoxia-induced synaptic disturbance was a marked increase in the frequency of sPSCs, which also began soon after the removal of oxygen and long before AD. This synaptic effect was accompanied by depletion of the readily releasable transmitter pools, as demonstrated by a decreased response to hyperosmotic solutions. The early [Ca2+]i rise, the early increase in transmitter release and the subsequent AD itself were all prevented by bathing in a cocktail containing blockers of ionotropic glutamate receptors. We found no evidence for involvement of pannexin hemichannels or TRPM7 channels in the early responses to hypoxia in this experimental preparation. Our data indicate that the earliest cellular consequences of cortical hypoxia are triggered by activation of glutamate-gated channels.

Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A1, A2A, A2B, and A3. Although adenosine exerts clear neuroprotective effects through A1 receptors during ischemia, the use of selective A1 receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A2B receptors in cerebral ischemia. This study explored the role of adenosine A2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A2Breceptor antagonism significantly prevented astrocyte modifications. Both A2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia.

Acute subdural hemorrhage (ASDH) is common and associated with severe morbidity and mortality. To date, the role of spontaneous cortical spreading depression (sCSD) in exaggerating secondary injury after ASDH, is poorly understood. The present study contains two experimental groups: First, we investigated and characterized the occurrence of sCSD after subdural blood infusion (300 μL) via tissue impedance (IMP) measurement in a rat model. Second, we compared the occurrence and influence of sCSD on lesion growth and neurological deficit in the presence and absence of whole blood constituents. In the first experimental group, three IMP traits could be distinguished after ASDH: no sCSD, recurrent sCSD, and constant elevated IMP (anoxic depolarization [AD]). In the second experimental group, sCSD occurred more often after autologous blood, compared with paraffin oil infusion. Lesion volume 7 days post-ASDH was 27.3 ± 6.8 mm3 after blood and 3.4 ± 2.1 mm3 after paraffin oil infusion. Subgroup analysis showed larger lesion size in animals with sCSD, than in those without. Further, occurrence of sCSD led to worse neurological outcomes in both groups. sCSD occurs early after ASDH and does not depend on the presence of whole blood constituents. However, numbers and degree of sCSD are more frequent and severe after autologous blood infusion, compared with an inert volume substance. The occurrence of sCSD leads to lesion growth and worse neurological outcome. Thus, our data advocate close monitoring and targeted treatment of sCSD after ASDH evacuation. [ABSTRACT FROM AUTHOR]

Highlights • Astrocytic responses to oxygen and glucose deprivation (OGD) were investigated in brain slice, an in vitro ischemia model. • OGD depolarized astrocytes in the Substantia Nigra and reduced GFAP immunoreactivity. • The membrane potential of nigral astrocytes did not repolarize during reperfusion (10–15 min). • In contrast, hippocampal astrocytes did not show acute ischemic sensitivity. • The ischemic impairment of nigral astrocyte function may contribute to sudden-onset vascular forms of parkinsonism. Abstract The Substantia Nigra is a brainstem nucleus critical for movement control. Although its dopamine-producing neurons degenerate in Parkinsons disease, little is known of the acute effects of ischemia in this region. We recently reported that oxygen and glucose deprivation (OGD) in brain slices, an in vitro ischemia model, evokes a profound depolarization and swelling of GABAergic neurons in the Substantia Nigra pars reticulata (SNr), but not dopaminergic neurons in the Substantia Nigra pars compacta (SNc). The current study characterised the effects of OGD on nigral astrocytes, and compared these with the established responses of astrocytes in the CA1 hippocampal region. Intracellular recordings were made from astrocytes at the border between SNc and SNr subregions, in midbrain slices from postnatal day 21–23 rats. Immunoreactivity for astrocyte-specific proteins was also assessed. OGD evoked a slow, then fast depolarization of nigral astrocytes. The fast phase developed during the anoxic depolarization (indicated by a fast negative shift of extracellular DC potential and increase in light transmittance) and rapid increase in extracellular K+ concentration in the SNr. This biphasic response resembled the OGD-evoked depolarization of hippocampal astrocytes. However, unlike the partial repolarization seen in hippocampal cells after reperfusion with O 2 and glucose, nigral astrocytes remained depolarized near 0 mV. In addition, immunoreactivity for glial fibrillary acidic protein-positive astrocytes markedly decreased in the Substantia Nigra after OGD, while in the hippocampus remained unchanged. These data indicate an acute post-ischemic withdrawal of astrocytic support in the Substantia Nigra, but not in the hippocampus. [ABSTRACT FROM AUTHOR]

Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A1, A2A, A2B, and A3. Although adenosine exerts clear neuroprotective effects through A1 receptors during ischemia, the use of selective A1 receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A2B receptors in cerebral ischemia. This study explored the role of adenosine A2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A2Breceptor antagonism significantly prevented astrocyte modifications. Both A2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A2B receptor subtype may thus represent a new class of neuroprotective drugs in ischemia. [ABSTRACT FROM AUTHOR]

The neocortex is highly susceptible to metabolic dysfunction. When exposed to global ischemia or anoxia, it suffers a slowly propagating wave of collective neuronal depolarization that ultimately impairs its structure and function. While the molecular signature of anoxic depolarization (AD) is well documented, little is known about the brain states that precede and follow AD onset. Here, by means of multisite extracellular local field potentials and intracellular recordings from identified pyramidal cells, we investigated the laminar expression of cortical activities induced by transient anoxia in rat primary somatosensory cortex. Soon after the interruption of brain oxygenation, we observed a well-organized sequence of stereotyped activity patterns across all cortical layers. This sequence included an initial period of beta-gamma activity, rapidly replaced by delta-theta oscillations followed by a decline in all spontaneous activites, marking the entry into a sustained period of electrical silence. Intracellular recordings revealed that cortical pyramidal neurons were depolarized and highly active during high-frequency activity, became inactive and devoid of synaptic potentials during the isoelectric state, and showed subthreshold composite synaptic depolarizations during the low-frequency period. Contrasting with the strong temporal coherence of pre-AD activities along the vertical axis of the cortical column, the onset of AD was not uniform across layers. AD initially occurred in layer 5 or 6 and then propagated bidirectionally in the upward and downward direction. Conversely, the post-anoxic waves that indicated the repolarization of cortical neurons upon brain reoxygenation did not exhibit a specific spatio-temporal profile. Pyramidal neurons from AD initiation site had a more depolarized resting potential and higher spontaneous firing rate compared to superficial cortical cells. We also found that the propagation pattern of AD was reliably reproduced by focal injection of an inhibitor of sodium‑potassium ATPases, suggesting that cortical AD dynamics could reflect layer-dependent variations in cellular metabolic regulations. [Display omitted] • Early anoxia is associated with stereotyped activities in all cortical layers. • Anoxic depolarization (AD) is initiated in layer 5 or 6. • Ouabain injection in deep layers replicates AD onset and spread. • A timely metabolic restoration can reverse AD. • Post-anoxic wave lacks a specific spatiotemporal profile. [ABSTRACT FROM AUTHOR]

Anoxic depolarization (AD) is a hallmark of ischemic brain damage. AD is associated with a spreading wave of neuronal depolarization and an increase in light transmittance. However, initiation and spread of AD across the layers of the somatosensory cortex, which is one of the most frequently affected brain regions in ischemic stroke, remains largely unknown. Here, we explored the initiation and propagation of AD in slices of the rat barrel cortex using extracellular local field potential (LFP) recordings and optical intrinsic signal (OIS) recordings. We found that ischemia-like conditions induced by oxygen-glucose deprivation (OGD) evoked AD, which manifested as a large negative LFP shift and an increase in light transmittance. AD typically initiated in one or more barrels and further spread across the entire slice with a preferential propagation through L4. Elevated extracellular potassium concentration accelerated the AD onset without affecting proneness of L4 to AD. In live slices, barrels were most heavily labeled by the metabolic level marker 2,3,5-triphenyltetrazolium chloride, suggesting that the highest metabolic demand is in L4 when compared to the other layers. Thus, L4 is the layer of the barrel cortex most prone to AD, which may be due to the highest metabolic demand and cell density in this layer.

During surgical treatment for cerebrovascular diseases, cortical hemodynamics are often controlled by bypass graft surgery, temporary occlusion of arteries, and surgical removal of veins. Since the brain is vulnerable to hypoxemia and ischemia, interruption of cerebral blood flow reduces the oxygen supply to tissues and induces irreversible damage to cells and tissues. Monitoring of cerebral hemodynamics and alteration of cellular structure during neurosurgery is thus crucial. Sequential recordings of red-green-blue (RGB) images of in vivo exposed rat brains were made during hyperoxia, normoxia, hypoxia, and anoxia. Monte Carlo simulation of light transport in brain tissue was used to specify relationships among RGB-values and oxygenated hemoglobin concentration (CHbO), deoxygenated hemoglobin concentration (CHbR), total hemoglobin concentration (CHbT), hemoglobin oxygen saturation (StO2), and scattering power b. Temporal courses of CHbO, CHbR, CHbT, and StO2 indicated physiological responses to reduced oxygen delivery to cerebral tissue. A rapid decrease in light scattering power b was observed after respiratory arrest, similar to the negative deflection of the extracellular direct current (DC) potential in so-called anoxic depolarization. These results suggest the potential of this method for evaluating pathophysiological conditions and loss of tissue viability. [ABSTRACT FROM AUTHOR]

Anoxic depolarization (AD) is a hallmark of ischemic brain damage. AD is associated with a spreading wave of neuronal depolarization and an increase in light transmittance. However, initiation and spread of AD across the layers of the somatosensory cortex, which is one of the most frequently affected brain regions in ischemic stroke, remains largely unknown. Here, we explored the initiation and propagation of AD in slices of the rat barrel cortex using extracellular local field potential (LFP) recordings and optical intrinsic signal (OIS) recordings. We found that ischemia-like conditions induced by oxygen-glucose deprivation (OGD) evoked AD, which manifested as a large negative LFP shift and an increase in light transmittance. AD typically initiated in one or more barrels and further spread across the entire slice with a preferential propagation through L4. Elevated extracellular potassium concentration accelerated the AD onset without affecting proneness of L4 to AD. In live slices, barrels were most heavily labeled by the metabolic level marker 2,3,5-triphenyltetrazolium chloride, suggesting that the highest metabolic demand is in L4 when compared to the other layers. Thus, L4 is the layer of the barrel cortex most prone to AD, which may be due to the highest metabolic demand and cell density in this layer. [ABSTRACT FROM AUTHOR]

Spreading depolarizations (SD) are waves of abrupt, near-complete breakdown of neuronal transmembrane ion gradients, are the largest possible pathophysiologic disruption of viable cerebral gray matter, and are a crucial mechanism of lesion development. Spreading depolarizations are increasingly recorded during multimodal neuromonitoring in neurocritical care as a causal biomarker providing a diagnostic summary measure of metabolic failure and excitotoxic injury. Focal ischemia causes spreading depolarization within minutes. Further spreading depolarizations arise for hours to days due to energy supply-demand mismatch in viable tissue. Spreading depolarizations exacerbate neuronal injury through prolonged ionic breakdown and spreading depolarization-related hypoperfusion (spreading ischemia). Local duration of the depolarization indicates local tissue energy status and risk of injury. Regional electrocorticographic monitoring affords even remote detection of injury because spreading depolarizations propagate widely from ischemic or metabolically stressed zones; characteristic patterns, including temporal clusters of spreading depolarizations and persistent depression of spontaneous cortical activity, can be recognized and quantified. Here, we describe the experimental basis for interpreting these patterns and illustrate their translation to human disease. We further provide consensus recommendations for electrocorticographic methods to record, classify, and score spreading depolarizations and associated spreading depressions. These methods offer distinct advantages over other neuromonitoring modalities and allow for future refinement through less invasive and more automated approaches. [ABSTRACT FROM AUTHOR]

Spreading depolarization (SD) is characterized by a massive redistribution of ions accompanied by an arrest in electrical activity that slowly propagates through neural tissue. It has been implicated in numerous human pathologies, including migraine, stroke, and traumatic brain injury, and thus the elucidation of control mechanisms underlying the phenomenon could have many health benefits. Here, we demonstrate the occurrence of SD in the brain of Drosophila melanogaster, providing a model system, whereby cellular mechanisms can be dissected using molecular genetic approaches. Propagating waves of SD were reliably induced by disrupting the extracellular potassium concentration ([K+]o), either directly or by inhibition of the Na+/K+-ATPase with ouabain. The disturbance was monitored by recording the characteristic surges in [K+]o using K+-sensitive microelectrodes or by monitoring brain activity by measuring direct current potential. With the use of wild-type flies, we show that young adults are more resistant to SD compared with older adults, evidenced by shorter bouts of SD activity and attenuated [K+]o disturbances. Furthermore, we show that the susceptibility to SD differs between wild-type flies and w1118 mutants, demonstrating that our ouabain model is influenced by genetic strain. Lastly, flies with low levels of protein kinase G (PKG) had increased latencies to onset of both ouabain-induced SD and anoxic depolarization compared with flies with higher levels. Our findings implicate the PKG pathway as a modulator of SD in the fly brain, and given the conserved nature of the signaling pathway, it could likely play a similar role during SD in the mammalian central nervous system. [ABSTRACT FROM AUTHOR]

The extent of anoxic depolarization (AD), the initial electrophysiological event during ischemia, determines the degree of brain region-specific neuronal damage. Neurons in higher brain regions exhibiting nonreversible, strong AD are more susceptible to ischemic injury as compared to cells in lower brain regions that exhibit reversible, weak AD. While the contrasting ADs in different brain regions in response to oxygen-glucose deprivation (OGD) is well established, the mechanism leading to such differences is not clear. Here we use computational modeling to elucidate the mechanism behind the brain region-specific recovery from AD. Our extended Hodgkin-Huxley (HH) framework consisting of neural spiking dynamics, processes of ion accumulation, and ion homeostatic mechanisms unveils that glial-vascular K clearance and Na/K-exchange pumps are key to the cell's recovery from AD. Our phase space analysis reveals that the large extracellular space in the upper brain regions leads to impaired Na/K-exchange pumps so that they function at lower than normal capacity and are unable to bring the cell out of AD after oxygen and glucose is restored. [ABSTRACT FROM AUTHOR]

Highlights: [•] Astrocytes exhibit characteristic changes in [Ca2+]i under OGD. [•] Astrocytic [Ca2+]i increase is synchronized with a neuronal anoxic depolarization. [•] Gap junctional couplings protect neurons as well as astrocytes during OGD. [ABSTRACT FROM AUTHOR]

Background and Purpose: Activation of α7 nicotinic acetylcholine receptors (nAChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type-II positive allosteric modulators (PAMs-II) of α7 nAChRs, such as 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea (PNU-120596). This proof-of-concept study presents a novel neuroprotective paradigm that converts endogenous choline/ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU-120596.Experimental Approach: An electrophysiological ex vivo cell injury assay (to quantify the susceptibility of hippocampal neurons to acute injury by complete oxygen and glucose deprivation; COGD) and an in vivo middle cerebral artery occlusion model of ischaemia were used in rats.Key Results: Choline (20-200 μM) in the presence, but not absence of 1 μM PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons, but not CA1 stratum radiatum interneurons, subjected to COGD in acute hippocampal slices and these effects were blocked by 20 nM methyllycaconitine, a selective α7 antagonist, thus, activation of α7 nAChRs was required. PNU-120596 alone was ineffective ex vivo. In in vivo experiments, both pre- and post-ischaemia treatments with PNU-120596 (30 mg·kg(-1) , s.c. and 1 mg·kg(-1) , i.v., respectively) significantly reduced the cortical/subcortical infarct volume caused by transient focal cerebral ischaemia. PNU-120596 (1 mg·kg(-1) , i.v., 30 min post-ischaemia) remained neuroprotective in rats subjected to a choline-deficient diet for 14 days prior to experiments.Conclusions and Implications: PNU-120596 and possibly other PAMs-II significantly improved neuronal survival in cerebral ischaemia by augmenting neuroprotective effects of endogenous choline/ACh. [ABSTRACT FROM AUTHOR]

Background and Purpose Activation of α7 nicotinic acetylcholine receptors (n AChRs) can be neuroprotective. However, endogenous choline and ACh have not been regarded as potent neuroprotective agents because physiological levels of choline/ ACh do not produce neuroprotective levels of α7 activation. This limitation may be overcome by the use of type- II positive allosteric modulators ( PAMs- II) of α7 n AChRs, such as 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)-urea ( PNU-120596). This proof-of-concept study presents a novel neuroprotective paradigm that converts endogenous choline/ ACh into potent neuroprotective agents in cerebral ischaemia by inhibiting α7 nAChR desensitization using PNU-120596. Experimental Approach An electrophysiological ex vivo cell injury assay (to quantify the susceptibility of hippocampal neurons to acute injury by complete oxygen and glucose deprivation; COGD) and an in vivo middle cerebral artery occlusion model of ischaemia were used in rats. Key Results Choline (20-200 μM) in the presence, but not absence of 1 μM PNU-120596 significantly delayed anoxic depolarization/injury of hippocampal CA1 pyramidal neurons, but not CA1 stratum radiatum interneurons, subjected to COGD in acute hippocampal slices and these effects were blocked by 20 nM methyllycaconitine, a selective α7 antagonist, thus, activation of α7 nAChRs was required. PNU-120596 alone was ineffective ex vivo. In in vivo experiments, both pre- and post-ischaemia treatments with PNU-120596 (30 mg·kg−1, s.c. and 1 mg·kg−1, i.v., respectively) significantly reduced the cortical/subcortical infarct volume caused by transient focal cerebral ischaemia. PNU-120596 (1 mg·kg−1, i.v., 30 min post-ischaemia) remained neuroprotective in rats subjected to a choline-deficient diet for 14 days prior to experiments. Conclusions and Implications PNU-120596 and possibly other PAMs- II significantly improved neuronal survival in cerebral ischaemia by augmenting neuroprotective effects of endogenous choline/ ACh. [ABSTRACT FROM AUTHOR]

Abstract: The hippocampus is comprised of two distinct subfields that show different responses to hypoxic-ischemic brain injury: the CA1 region is particularly susceptible whereas the dentate gyrus (DG) is quite resistant. Our aim was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of A2A adenosine receptor antagonism to recovery of synaptic activity after OGD. Extracellular recordings of field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG in brain slices prepared from male Wistar rats were used. A 9-min OGD is needed in the DG to always induce the appearance of anoxic depolarization (AD) and the irreversible block of synaptic activity, as recorded up to 24 h from the end of the insult, whereas only 7-min OGD is required in the CA1 region. Selective antagonism of A2A adenosine receptors by ZM241385 significantly prevents or delays the appearance of AD and protects from the irreversible block of neurotransmission induced by 9-min OGD in the DG. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone of DG in slices prepared from 5-bromo-2′-deoxyuridine (BrdU) treated rats was investigated. Slices were further incubated with an immature neuronal marker, doublecortin (DCX). The number of BrdU+ cells was significantly decreased 6 h after 9-min OGD and this effect was antagonized by ZM241385. After 24 h from the end of 9-min OGD, the number of BrdU+ cells returned to that found before OGD and increased arborization of tertiary dendrites of DCX+ cells was observed. The adenosine A2A antagonist ZM241385 protects from synaptic failure and from decreased proliferation of immature neuronal cells at a precocious time after OGD. [Copyright &y& Elsevier]

Abstract: Current responses from CA1 neurons and stratum oriens astrocytes were recorded from hippocampal brain slices by means of the whole-cell patch-clamp technique. Anoxic depolarization (AD) was induced by an oxygen/glucose-deprived (OGD) medium also containing sodium iodoacetate and antimycin, in order to block glycolysis and oxidative phosphorylation, respectively. Anoxic depolarization has been reported to be due to the sudden increase of the extracellular K+ concentration and the accompanying explosive rise in glutamate concentration. We asked ourselves whether the release of ATP activating P2X7 receptors is also involved in the AD. Although, the AD was evoked in absolute synchrony in neurons and astrocytes, and the NMDA receptor antagonistic AP-5 depressed these responses, neither the non-selective P2 receptor antagonist PPADS, nor the highly selective P2X7 receptor antagonist A438079 interfered with the AD or its delay time in neurons/astrocytes after inducing chemical hypoxia. However, A438079, but not PPADS increased in astrocytes the slow inward current observed in a hypoxic medium. It is concluded that ATP co-released with glutamate by hypoxic stimulation has only a minor function in the present brain slice system. [Copyright &y& Elsevier]

To investigate the role of purinergic P2 receptors under ischemia, we studied the effect of P2 receptor antagonists on synaptic transmission and mitogen-activated protein kinase (MAPK) activation under oxygen and glucose deprivation (OGD) in rat hippocampal slices. The effect of the P2 antagonists pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate (PPADS, unselective, 30 μ), N-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179, selective for P2Y receptor, 10 μ), Brilliant Blue G (BBG, selective for P2X receptor, 1 μ), and 5-[[[(3-phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid (A-317491, selective for P2X receptor, 10 μ), and of the newly synthesized P2X receptor antagonists 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)adenine (PX21, 1 μ) and 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)- N-methyladenine (PX24, 1 μ), on the depression of field excitatory postsynaptic potentials (fEPSPs) and anoxic depolarization (AD) elicited by 7 min of OGD were evaluated. All antagonists significantly prevented these effects. The extent of CA1 cell injury was assessed 3 h after the end of 7 min of OGD by propidium iodide staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 μ), MRS2179 (10 μ), and BBG (1 μ). Western blot analysis showed that, 10 min after the end of the 7 min of OGD, extracellular signal-regulated kinase (ERK)1/2 MAPK activation was significantly increased. MRS2179, BBG, PPADS and A-317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors 1,4-diamino-2,3-dicyano-1,4- bis(2-aminophenylthio)butadiene (U0126, 10 μ) and α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile (SL327, 10 μ) showed significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in neuronal damage induced by OGD. These results indicate that subtypes of hippocampal P2 purinergic receptors have a harmful effect on neurotransmission in the CA1 hippocampus by participating in AD appearance and activation of ERK1/2. [ABSTRACT FROM AUTHOR]

Abstract: A new remote-controlled interface-type chamber was designed in order to conduct experiments in brain slices involving gas, fluid, and temperature changes with as little tissue manipulation as possible. The chamber allows for extremely quick changes between different fluid and/or gaseous phases and for active cooling as well as heating by using a set of electromechanical valves and Peltier elements. The design drawings are complemented by exemplary tests of temperature and gas changes, and electrophysiological recordings of slices manipulated with gas and fluid alterations were used to test the efficacy and accuracy of the design. Changing between normoxia and anoxia needs less than 30s, while the readjustment of the chamber to a new, preset temperature is accomplished in about 1min. Supplementary data provide a proposal for the electronic circuit diagram. This chamber design should simplify data acquisition in interface environments. [ABSTRACT FROM AUTHOR]

Abstract: Current in vitro models of stroke involve applying oxygen-glucose deprived (OGD) media over an entire brain slice or plate of cultured neurons. Thus, these models fail to mimic the focal nature of stroke as observed clinically and with in vivo rodent models of stroke. Our aim was to develop a novel in vitro brain slice model of stroke that would mimic focal ischemia and thus allow for the investigation of events occurring in the penumbra. This was accomplished by focally applying OGD medium to a small portion of a brain slice while bathing the remainder of the slice with normal oxygenated media. This technique produced a focal infarct on the brain slice that increased as a function of time. Electrophysiological recordings made within the flow of the OGD solution (“core”) revealed that neurons rapidly depolarized (anoxic depolarization; AD) in a manner similar to that observed in other stroke models. Edaravone, a known neuroprotectant, significantly delayed this onset of AD. Electrophysiological recordings made outside the flow of the OGD solution (“penumbra”) revealed that neurons within this region progressively depolarized throughout the 75min of OGD application. Edaravone attenuated this depolarization and doubled neuronal survival. Finally, synaptic transmission in the penumbra was abolished within 50min of focal OGD application. These results suggest that this in vitro model mimics events that occur during focal ischemia in vivo and can be used to determine the efficacy of therapeutics that target neuronal survival in the core and/or penumbra. [Copyright &y& Elsevier]

Abstract: Measurement of intrinsic optical signals (IOSs) is an attractive technique for monitoring tissue viability in brains since it enables noninvasive, real-time monitoring of morphological characteristics as well as physiological and biochemical characteristics of tissue. We previously showed that light scattering signals reflecting cellular morphological characteristics were closely related to the IOSs associated with the redox states of cytochrome c oxidase in the mitochondrial respiratory chain. In the present study, we examined the relationship between light scattering and energy metabolism. Light scattering signals were transcranially measured in rat brains after oxygen and glucose deprivation, and the results were compared with concentrations of cerebral adenosine triphosphate (ATP) measured by luciferin–luciferase bioluminescence assay. Electrophysiological signal was also recorded simultaneously. After starting saline infusion, EEG activity ceased at 108±17s, even after which both the light scattering signal and ATP concentration remained at initial levels. However, light scattering started to change in three phases at 236±15s and then cerebral ATP concentration started to decrease at about 260s. ATP concentration significantly decreased during the triphasic scattering change, indicating that the start of scattering change preceded the loss of cerebral ATP. The mean time difference between the start of triphasic scattering change and the onset of ATP loss was about 24s in the present model. DC potential measurement showed that the triphasic scattering change was associated with anoxic depolarization. These findings suggest that light scattering signal can be used as an indicator of loss of tissue viability in brains. [Copyright &y& Elsevier]

Background and Purpose: Activation of adenosine A(2A) receptors in the CA1 region of rat hippocampal slices during oxygen-glucose deprivation (OGD), a model of cerebral ischaemia, was investigated.Experimental Approach: We made extracellular recordings of CA1 field excitatory postsynaptic potentials (fepsps) followed by histochemical and immunohistochemical techniques coupled to Western blots.Key Results: OGD (7 or 30 min duration) elicited an irreversible loss of fepsps invariably followed by the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. The application of the selective adenosine A(2A) receptor antagonist, ZM241385 (4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino]ethyl)phenol; 100-500 nmolxL(-1)) prevented or delayed AD appearance induced by 7 or 30 min OGD and protected from the irreversible fepsp depression elicited by 7 min OGD. Two different selective adenosine A(2A) receptor antagonists, SCH58261 and SCH442416, were less effective than ZM241385 during 7 min OGD. The extent of CA1 cell injury was assessed 3 h after the end of 7 min OGD by propidium iodide. Substantial CA1 pyramidal neuronal damage occurred in untreated slices, exposed to OGD, whereas injury was significantly prevented by 100 nmolxL(-1) ZM241385. Glial fibrillary acid protein (GFAP) immunostaining showed that 3 h after 7 min OGD, astrogliosis was appreciable. Western blot analysis indicated an increase in GFAP 30 kDa fragment which was significantly reduced by treatment with 100 nmolxL(-1) ZM241385.Conclusions and Implications: In the CA1 hippocampus, antagonism of A(2A) adenosine receptors by ZM241385 was protective during OGD (a model of cerebral ischaemia) by delaying AD appearance, decreasing astrocyte activation and improving neuronal survival. [ABSTRACT FROM AUTHOR]

Abstract: While the vasomotor effects of pCO2 modulation are well documented, the influence of the carbon dioxide–bicarbonate system on the ischemia tolerance of brain tissue itself is controversial. Guinea-pig hippocampal tissue was subjected to ischemia simulation in an interface environment and examined electrophysiologically. Characteristics of anoxic depolarization as well as the postischemic recovery of evoked potentials were registered. During ischemia simulation, pH was changed and afterwards restored to 7.4. pH of 7.6 (n=6), and 7.8 (n=6) were adjusted by increasing bicarbonate concentration without changing pCO2, while pH 8.2 was reached either with normal pCO2 (n=8) or with zero CO2 (n=9). pH 7.1 was created by doubling pCO2 (n=22) or reducing bicarbonate (n=21), while acid pH of 6.9 (high pCO2 and low bicarbonate) led to erratic measurements in the interface setup. Alkalotic conditions did not improve electrophysiological stability of the tissue, and pH 8.2 impeded the recovery of evoked potentials. Hypercarbic pH 7.1 led to significantly longer latency of depolarization while the same pH with lowered bicarbonate did not. Evoked potentials, however, recovered only partially after ischemia at hypercarbic pH 7.1. Once the tissue had recovered from anoxic depolarization at control pH, hypercarbic acidosis did not have any further protective effect when ischemia simulation was repeated (n=12). These results do not strengthen the concept of hyperventilation in intensive care, while they suggest a potential of hypercarbia within broader strategies delaying the onset of secondary brain damage. [Copyright &y& Elsevier]

δ-Opioid receptor (DOR) activation is neuroprotective against short-term anoxic insults in the mammalian brain. This protection may be conferred by inhibition of N-methyl-D-aspartate receptors (NMDARs), whose over-activation during anoxia otherwise leads to a deleterious accumulation of cytosolic calcium ([Ca2+]c), severe membrane potential (Em) depolarization and excitotoxic cell death (ECD). Conversely, NMDAR activity is decreased by -50% with anoxia in the cortex of the painted turtle, and large elevations in [Ca2+]c, severe Em depolarization and ECD are avoided. DORs are expressed in high quantity throughout the turtle brain relative to the mammalian brain; however, the role of DORs in anoxic NMDAR regulation has not been investigated in turtles. We examined the effect of DOR blockade with naltrindole (1-10μ.mol l-1) on Em, NMDAR activity and [Ca2+]c homeostasis in turtle cortical neurons during normoxia and the transition to anoxia. Naltrindole potentiated normoxic NMDAR currents by 78±5% and increased [Ca2+]c by 13±4%. Anoxic neurons treated with naltrindole were strongly depolarized, NMDAR currents were potentiated by 70±15%, and [Ca2+]c increased 5-fold compared with anoxic controls. Following naltrindole washout, Em remained depolarized and [Ca2+]c became further elevated in all neurons. The naltrindole-mediated depolarization and increased [Ca2+]c were prevented by NMDAR antagonism or by perfusion of the Gi protein agonist mastoparan-7, which also reversed the naltrindole-mediated potentiation of NMDAR currents. Together, these data suggest that DORs mediate NMDAR activity in a Gi-dependent manner and prevent deleterious NMDAR-mediated [Ca2+]c influx during anoxic insults in the turtle cortex. [ABSTRACT FROM AUTHOR]

Abstract: Neuronal membrane potential (E m) regulates the activity of excitatory voltage-sensitive channels. Anoxic insults lead to a severe loss of E m and excitotoxic cell death (ECD) in mammalian neurons. Conversely, anoxia-tolerant freshwater turtle neurons depress energy usage during anoxia by altering ionic conductance to reduce neuronal excitability and ECD is avoided. This wholesale alteration of ion channel and pump activity likely has a significant effect on E m. Using the whole-cell patch clamp technique we recorded changes in E m from turtle cortical neurons during a normoxic to anoxic transition in the presence of various ion channel/pump modulators. E m did not change with normoxic perfusion but underwent a reversible, mild depolarization of 8.1±0.2 mV following anoxic perfusion. This mild anoxic depolarization (MAD) was not prevented by the manipulation of any single ionic conductance, but was partially reduced by pre-treatment with antagonists of GABAA receptors (5.7±0.5 mV), cellular bicarbonate production (5.3±0.2 mV) or K+ channels (6.0±0.2 mV), or by perfusion of reactive oxygen species scavengers (5.2±0.3 mV). Furthermore, all of these treatments induced depolarization in normoxic neurons. Together these data suggest that the MAD may be due to the summation of numerous altered ion conductance states during anoxia. [Copyright &y& Elsevier]

Abstract: This study shows that two whole isolated preparations from the young mouse, the neocortical ‘slab’ and the hippocampal formation, are useful for imaging studies requiring both global monitoring using light transmittance (LT) imaging and high resolution cellular monitoring using 2-photon laser scanning microscopy (2PLSM). These preparations share advantages with brain slices such as maintaining intrinsic neuronal properties and avoiding cardiac or respiratory movement. Important additional advantages include the maintenance of all local input and output pathways, the absence of surfaces injured by slicing and the preservation of three-dimensional tissue structure. Using evoked extracellular field recording, we demonstrate long-term (hours) viability of both whole preparations. We then show that propagating cortical events such as anoxic depolarization (AD) and spreading depression (SD) can be imaged in both preparations, yielding results comparable to those in brain slices but retaining the tissue''s three-dimensional structure. Using transgenic mice expressing green fluorescent protein (GFP) in pyramidal and granule cell neurons, 2PLSM confirms that these preparations are free of the surface damage observed in sliced brain tissue. Moreover the neurons undergo swelling with accompanying dendritic beading following AD induced by simulated ischemia, similar to cortical damage described in vivo. [Copyright &y& Elsevier]

Abstract: This study was designed to characterize nitric oxide (NO) production and anoxic depolarization in the rat hippocampus during transient forebrain ischemia using two NO synthase (NOS) inhibitors, l-N 5-(1-iminoethyl)ornithine (l-NIO), a relatively selective endothelial NOS (eNOS) inhibitor, and 7-nitroindazole, a relatively selective neuronal NOS (nNOS) inhibitor, and an NO scavenger, [2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] (carboxy-PTIO). We measured the mean arterial blood pressure, hippocampal blood flow, NO concentration and direct current potential before, during and after transient forebrain ischemia, which was induced by 4-vessel occlusion for 10 min. Saline, l-NIO (20 mg/kg), 7-nitroindazole (25 mg/kg), l-NIO (20 mg/kg)+7-nitroindazole (25 mg/kg) or carboxy-PTIO (1 mg/kg) was administered intraperitoneally 20 min before the onset of ischemia. We observed early and sharp NO production in the hippocampus during ischemia in the saline group. This NO increase during ischemia was significantly reduced by l-NIO (20 mg/kg)+7-nitroindazole (25 mg/kg) or carboxy-PTIO (1 mg/kg), but not l-NIO (20 mg/kg) or 7-nitroindazole (25 mg/kg). On the other hand, NO production after ischemia was significantly reduced by 7-nitroindazole (25 mg/kg), l-NIO (20 mg/kg)+7-nitroindazole (25 mg/kg) or carboxy-PTIO (1 mg/kg), but not l-NIO (20 mg/kg). The peak latency of NO production during ischemia always preceded the onset latency of anoxic depolarization in both the saline group and the carboxy-PTIO group. In the carboxy-PTIO group, the onset latency of anoxic depolarization was significantly longer than that in the saline group. Moreover, carboxy-PTIO significantly reduced the anoxic depolarization amplitude, compared with that of the saline group. These results suggest that both NOS-dependent and-independent NO formation contributes to early and sharp NO production during ischemia, and that this NO increase is, at least in part, related to the triggering of anoxic depolarization. [Copyright &y& Elsevier]

Ischemic depolarizing events, such as repetitive spontaneous periinfarct spreading depolarizations (PIDs), expand the infarct size after experimental middle cerebral artery (MCA) occlusion. This worsening may result from increased metabolic demand, exacerbating the mismatch between cerebral blood flow (CBF) and metabolism. Here, we present data showing that anoxic depolarization (AD) and PIDs caused vasoconstriction and abruptly reduced CBF in the ischemic cortex in a distal MCA occlusion model in mice. This reduction in CBF during AD increased the area of cortex with 20% or less residual CBF by 140%. With each subsequent PID, this area expanded by an additional 19%. Drugs that are known to inhibit cortical spreading depression (CSD), such as N-methyl-D-aspartate receptor antagonists MK-801 and 7-chlorokynurenic acid, and σ-1 receptor agonists dextromethorphan and carbetapentane, did not reduce the frequency of PIDs, but did diminish the severity of episodic hypoperfusions, and prevented the expansion of severely hypoperfused cortex, thus improving CBF during 90 mins of acute focal ischemia. In contrast, AMPA receptor antagonist NBQX, which does not inhibit CSD, did not impact the deterioration in CBF. When measured 24 h after distal MCA occlusion, infarct size was reduced by MK-801, but not by NBQX. Our results suggest that AD and PIDs expand the CBF deficit, and by so doing negatively impact lesion development in ischemic mouse brain. Mitigating the vasoconstrictive neurovascular coupling during intense ischemic depolarizations may provide a novel hemodynamic mechanism of neuroprotection by inhibitors of CSD.Journal of Cerebral Blood Flow & Metabolism (2006) 26, 1018–1030. doi:10.1038/sj.jcbfm.9600252; published online 7 December 2005 [ABSTRACT FROM AUTHOR]

During brain anoxia or ischemia, a decrease in the level of ATP leads to a sudden decrease in transmembrane ion gradients [anoxic depolarization (AD)]. This releases glutamate by reversing the operation of glutamate transporters, which triggers neuronal death. By whole-cell clamping CA1 pyramidal cells, we investigated the energy stores that delay the occurrence of the AD in hippocampal slices when O2 and glucose are removed. With glycolytic and mitochondrial ATP production blocked in P12 slices, the AD occurred in ∼7 min at 33°C, reflecting the time needed for metabolic activity to consume the existing ATP and phosphocreatine, and for subsequent ion gradient decrease. Allowing glycolysis fueled by glycogen, in the absence of glucose, delayed the AD by 5.5 min, whereas superfused glucose prevented the AD for > 1 h. With glycolysis blocked, the latency to the AD was 6.5 min longer when mitochondria were allowed to function, demonstrating that metabolites downstream of glycolysis (pyruvate, citric acid cycle intermediates, and amino acid oxidation) provide a significant energy store for oxidative phosphorylation. With glycolysis blocked but mitochondria functioning, superfusing lactate did not significantly delay the AD, showing that ATP production from lactate is much less than that from endogenous metabolites. These data demonstrate a preferential role for glycolysis in preventing the AD. They also define a hierarchy of pool sizes for hippocampal energy stores and suggest that brain ATP production from glial lactate may not be significant in conditions of energy deprivation. [ABSTRACT FROM AUTHOR]

We investigated the dose–response relationship of protection by creatine against ischemic damage, and we asked whether or not such protection may be observed in invertebrate neurons that might provide a simpler experimental model. Rat isolated pyramidal neurons from the CA3 region of hippocampus subjected to ischemia (“in vitro ischemia”) showed anoxic depolarization (AD) after 3–7 min of incubation in anoxic medium. Membrane potential (MP) was reduced 25–78% from preanoxic value. Inward current was decreased by an average 49%. Supplementation with creatine protected against these changes, with 1 mM being the minimal effective concentration, 2 mM providing a near-maximal protection, a maximal effect being obtained with 5 mM creatine. No additional protection was provided by up to 20 mM creatine. Isolated giant neurons of Lymnaea stagnalis showed a similar response to in vitro ischemia. However, a clear seasonal dependence of sensitivity of these cells was detected. In cells obtained during summertime (May–August), AD latency ranged from 3 to 10 min; during wintertime (December–March), this response did not occur even after 25–50 min. The addition of creatine to the medium did not cause changes in AD latency, probably because these neurons rely on a phosphoarginine/arginine energy system. However, treatment of the cells, harvested during summertime, with 2 mM arginine did provide clear protection against anoxic–aglycaemic changes. Summing up, besides confirming previous findings on creatine protection in mammalian neurons, we (1) better characterized their dose–response relationship and extended the findings to the CA3 region and to isolated neurons, (2) found that invertebrate neurons are not protected by creatine but by arginine supplementation and (3) reported a novel mechanism of seasonal dependence in sensitivity of in vitro ischemia by invertebrate neurons. [Copyright &y& Elsevier]

To clarify the functions of nitric oxide (NO) induced by either neuronal NO synthase (nNOS) or endothelial NO synthase (eNOS) after transient cerebral ischemia, we investigated the effects of l-N5-(1-iminoethyl)ornithine (l-NIO), a relatively selective eNOS inhibitor, and 7-nitroindazole (7-NI), a relatively selective nNOS inhibitor, on hippocampal dysfunction caused by cerebral ischemia. We measured mean arterial blood pressure (MABP), hippocampal blood flow, direct current (DC) potential, CA1 population spike (PS) and extracellular concentrations of glutamate from rat hippocampus after transient forebrain ischemia, which was induced by four-vessel occlusion for 10 min. l-NIO (20 mg/kg) and 7-NI (25 mg/kg) were administered intraperitoneally 20 min before ischemia. l-NIO, but not 7-NI, increased MABP before, during and after ischemia, compared with the vehicle group. 7-NI, but not l-NIO, reduced the amplitude of anoxic depolarization induced by ischemia. 7-NI recovered the PS amplitude in part 60 min after ischemia. 7-NI, but not l-NIO, reduced the ischemia-induced levels of glutamate. These results indicate that nNOS inhibition with 7-NI improves, at least in part, hippocampal dysfunction after ischemia, while eNOS inhibition with l-NIO worsens it. [Copyright &y& Elsevier]

Global cerebral ischemia models for genetically engineered mice are of particular importance for the study of delayed neuronal death, but have been complicated by variability of vascular anatomy. Here we developed a 5-min cardiac arrest model that was not affected by vascular anatomy, and evaluated the hippocampal neuronal injury in BL/6 and SV129 mice. Despite prolonged anoxic depolarization for approximately 7 min, however, no consistent ischemic neuronal injury was noted in the CA1 sector of the hippocampus in both strains. Thus, our observations suggested that murine hippocampal neurons are relatively resistant to ischemia compared with those in other rodents. [Copyright &y& Elsevier]

Abstract Glutamate release in ischaemia triggers neuronal death. The major glial glutamate transporter, GLT-1, might protect against glutamate-evoked death by removing extracellular glutamate, or contribute to death by reversing and releasing glutamate. Previous studies of the role of GLT-1 in ischaemia have often used the GLT-1 blocker dihydrokainate at concentrations that affect transporters other than GLT-1 and which affect kainate, N -methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In hippocampal slices from postnatal day 14 mice lacking GLT-1, the current response of area CA1 pyramidal cells to superfused AMPA and NMDA (which are not taken up) was unaffected, whereas the response to 100 µm glutamate was more than doubled relative to that in wild-type littermates, a finding consistent with a decrease in glutamate uptake. In response to a few minutes of simulated ischaemia, pyramidal cells in wild-type mice showed a large and sudden inward glutamate-evoked current [the anoxic depolarization (AD) current], which declined to a less inward plateau. In mice lacking GLT-1, the time to the occurrence of the AD current, its amplitude, the size of the subsequent plateau current and the block of the plateau current by glutamate receptor blockers were all indistinguishable from those in wild-type mice. We conclude that GLT-1 does not contribute significantly to glutamate release or glutamate removal from the extracellular space in early simulated ischaemia. These data are consistent with glutamate release being by reversal of neuronal transporters, and with uptake into glia being compromised by the ischaemia-evoked fall in the level of ATP needed to convert glutamate into glutamine. [ABSTRACT FROM AUTHOR]

Therapeutic hypothermia is now well established to significantly improve survival without disability after neonatal encephalopathy (NE). To further improve outcomes, we need to better understand the mechanisms of brain injury. The central finding, which offers the potential for neuroprotective and neurorestorative interventions, is that brain damage after perinatal hypoxia-ischemia evolves slowly over time. Although brain cells may die during profound hypoxia-ischemia, even after surprisingly severe insults many cells show transient recovery of oxidative metabolism during a "latent" phase characterized by actively suppressed neural metabolism and activity. Critically, after moderate to severe hypoxia-ischemia, this transient recovery is followed after ~6 h by a phase of secondary deterioration, with delayed seizures, failure of mitochondrial function, cytotoxic edema, and cell death over ~72 h. This is followed by a tertiary phase of remodeling and recovery. This review discusses the mechanisms of injury that occur during the primary, latent, secondary and tertiary phases of injury and potential treatments that target one or more of these phases. By analogy with therapeutic hypothermia, treatment as early as possible in the latent phase is likely to have the greatest potential to prevent injury ("neuroprotection"). In the secondary phase of injury, anticonvulsants can attenuate seizures, but show limited neuroprotection. Encouragingly, there is now increasing preclinical evidence that late, neurorestorative interventions have potential to improve long-term outcomes. [ABSTRACT FROM AUTHOR]