Ultrastructural Changes in the Frog Brain in the Presence of High Concentrations of Glutamate and an NO-Generating Compound

This review summarizes published data and original findings on the morphology of neurons and glial cells in normal conditions and model conditions that simulate those in the human brain during stroke. Ultrastructural changes that occur in the presence of high concentrations of glutamate (Glu, 0.1–5.0 M) and the NO-generating compound NaNO₂ (0.1–5.0 mM) were studied in the cerebella (frog cerebellum), which is one of the simplest circuitries structurally. Such studies and data analyses are important because hyperstimulation of Glu receptors is a leading pathogenetic factor of neuronal damage during a stroke. High Glu concentrations exert a toxic effect and damage cerebellar neurons and glial cells. Mitochondria are de-energized, ionic homeostasis is distorted, the intracellular Ca²⁺ concentration increases, and constitutive NO synthases are activated in the process. The changes result in an increase in the contents of NO and its transformation products, which are involved in a negative feedback mechanism from postsynaptic neurons to presynaptic cells. Biochemical processes are consequently affected, and morphological changes are induced in neurons and glial cells, leading to their swelling. At the same time, ultrastructural compensatory adaptive mechanisms develop to reduce the damaging effect of high concentrations of Glu and NO-generating compounds.     

Extracellular Dopamine and Serotonin in the Rat Striatum During Transient Ischaemia of Different Severities: A Microdialysis Study

Abstract: Generalised neurotransmitter overflow into the extracellular space on cerebral ischaemia has been widely reported and implicated in events leading to subsequent neu-ronal death. As little is known about the effect of depth of ischaemia on these changes, we have subjected anaesthetised rats to a sequence of four challenges [high K⁺ stimulus, moderate (penumbral) ischaemia, severe ischaemia, cardiac arrest] and have concurrently monitored both electrophysio-logical parameters and changes in extracellular dopamine, serotonin, and their metabolites in the striatum. Oi'particu-lar relevance to human stroke therapy was penumbral ischaemia, where ionic homeostasis was maintained even though electrical function was lost. All challenges increased extracellular monoamines, although levels were significantly greater when ischaemia was severe enough to produce sustained anoxic depolarisation. Baseline levels were rapidly restored during recovery phases. Acidic monoamine metabolites decreased significantly during each insult, returning to basal levels during reperfusion after moderate ischaemia, and to significantly higher levels after severe ischaemia. Results indicate that sustained anoxic depolarisation may be a critical factor in determining outcome after ischaemia, being associated with significantly greater release of monoamines, and impairment of electrical function recovery.     

Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K⁺ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (V_m), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in V_m. Cell volume and V_m changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by V_m depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K⁺ or Cl⁻ channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in V_m– define the success of RVD.     

Differential Response of Neural Cells to Trauma-Induced Swelling In Vitro

Brain edema and the associated increase in intracranial pressure are major consequences of traumatic brain injury (TBI) that accounts for most early deaths after TBI. We recently showed that acute severe trauma to cultured astrocytes results in cell swelling. We further examined whether trauma induces cell swelling in neurons and microglia. We found that severe trauma also caused cell swelling in cultured neurons, whereas no swelling was observed in microglia. While severe trauma caused cell swelling in both astrocytes and neurons, mild trauma to astrocytes, neurons, and microglia failed to cell swelling. Since extracellular levels of glutamate are increased in brain post-TBI and microglia are known to release cytokine, and direct exposure of astrocytes to these molecules are known to stimulate cell swelling, we examined whether glutamate or cytokines have any additive effect on trauma-induced cell swelling. Exposure of cultured astrocytes to trauma caused cell swelling, and such swelling was potentiated by the exposure of traumatized astrocytes to glutamate and cytokines. Conditioned medium (CM) from traumatized astrocytes had no effect on neuronal swelling post-trauma, while CM from traumatized neurons and microglia potentiated the effect of trauma on astrocyte swelling. Further, trauma significantly increased the Na–K–Cl co-transporter (NKCC) activity in neurons, and that inhibition of NKCC activity diminished the trauma-induced neuronal swelling. Our results indicate that a differential sensitivity to trauma-induced cell swelling exists in neural cells and that neurons and microglia are likely to be involved in the potentiation of the astrocyte swelling post-trauma.     

Vinpocetine regulates cation channel permeability of inner retinal neurons in the ischaemic retina

Vinpocetine is a natural drug which exerts neuroprotective effects in ischaemia of the brain through actions on cation channels, glutamate receptors and other pathways. This study investigated the effect of vinpocetine on cation channel permeability of inner retinal neurons after acute retinal metabolic insult. We focused on amacrine and ganglion cells immunoreactive for calretinin or parvalbumin due to their previously documented susceptibility to ischaemia. Using the probe, 1-amino-4-guanidobutane (AGB), we observed increased cation channel permeability across amacrine and ganglion cells under ischaemia and hypoglycaemia but not anoxia. Calretinin and parvalbumin immunoreactivity was also reduced during ischaemia and hypoglyacemia but not anoxia. Vinpocetine decreased AGB entry into ischaemic and hypoglycaemic ganglion cells indicating that the drug can modulate unregulated cation entry. In addition, vinpocetine prevented the loss of calretinin and parvalbumin immunoreactivity following ischaemia suggesting it may indirectly regulate intracellular calcium. Vinpocetine also reduced AGB permeability in selected amacrine and ganglion cell populations following N-methyl-D-aspartate (NMDA) but not kainate activation suggesting that vinpocetine’s regulation of cation channel permeability may partly involve NMDA sensitive glutamate receptors.     

Glial cell-derived glutamate mediates autocrine cell volume regulation in the retina: activation by VEGF

Astroglial cells are a source for gliotransmitters such as glutamate and ATP. We demonstrate here that gliotransmitters have autocrine functions in the regulation of cellular volume. Hypoosmotic stress in the presence of inflammatory mediators or oxidative stress, and during blockade or down-regulation of potassium channels, induces swelling of retinal glial cells. Vascular endothelial growth factor inhibits the osmotic swelling of glial cells in retinal slices or isolated cells. This effect was mediated by a kinase domain region/flk-1 receptor-evoked calcium dependent release of glutamate from glial cells, and subsequent stimulation of glial group I/II metabotropic glutamate receptors. Activation of kinase domain region/flk-1 or glutamate receptors evoked an autocrine swelling-inhibitory purinergic signaling cascade that was calcium-independent. This cascade involved the release of ATP and adenosine, and the activation of purinergic P2Y₁ and adenosine A1 receptors, resulting in the opening of potassium and chloride channels and inhibition of cellular swelling. The glutamatergic-purinergic regulation of the glial cell volume may be functionally important in the homeostasis of the extracellular space volume during intense neuronal activation which is associated with a swelling of neuronal cell structures in the retina. However, glial cell-derived glutamate may also contribute to the swelling of activated neurons since metabolic poisoning of glial cells by iodoacetate inhibits the neuronal cell swelling mediated by activation of ionotropic glutamate receptors.     

Adrenomedullin protects neurons against oxygen glucose deprivation stress in an autocrine and paracrine manner

The understanding of mechanisms involved in ischaemic brain tolerance may provide new therapeutical targets for stroke. In vivo genomic studies revealed an up-regulation of adrenomedullin expression by hypoxic pre-conditioning. Furthermore, adrenomedullin reduced ischaemia-induced brain damage in rodents. However, whether adrenomedullin is involved in hypoxic pre-conditioning-induced tolerance and whether adrenomedullin protects directly neurons against ischaemia remain unknown. Using a neuronal model of hypoxic pre-conditioning and oxygen glucose deprivation (OGD), we showed that 0.1% or 0.5% of O₂ pre-conditioning reduced the OGD-induced neuronal death, whereas 1% or 2% of O₂ pre-treatment did not induce neuroprotection. Adrenomedullin expression increased following the hypoxic period, and following OGD only in pre-conditioned (0.1% or 0.5% of O₂) neurons. Adrenomedullin pre-treatment and post-treatment reduced the OGD-induced neuronal death, partly through PI3kinase-dependent pathway. However, adrenomedullin antagonism during hypoxic pre-conditioning failed to inhibit the neuroprotection whereas adrenomedullin antagonism following OGD abolished the hypoxic pre-conditioning-induced neuroprotection. Finally, we showed that adrenomedullin is involved in neuroprotection induced by endothelial cells and microglia. In contrast, neuroprotection induced by astrocytes occurred through adrenomedullin-independent mechanisms. Altogether, our results suggest that adrenomedullin is an effector of the hypoxic pre-conditioning-induced neuronal tolerance and a potent autocrine and paracrine neuroprotective factor during cerebral ischaemia.     

In Silico Study of the Influence of Intensity and Duration of Blood Flow Reduction on Cell Death Through Necrosis or Apoptosis During Acute Ischemic Stroke

Ischemic stroke involves numerous and complex pathophysiological mechanisms including blood flow reduction, ionic exchanges, spreading depressions and cell death through necrosis or apoptosis. We used a mathematical model based on these phenomena to study the influences of intensity and duration of ischemia on the final size of the infarcted area. This model relies on a set of ordinary and partial differential equations. After a sensibility study, the model was used to carry out in silico experiments in various ischemic conditions. The simulation results show that the proportion of apoptotic cells increases when the intensity of ischemia decreases, which contributes to the model validation. The simulation results also show that the influence of ischemia duration on the infarct size is more complicated. They suggest that reperfusion is beneficial when performed in the early stroke but may be either inefficacious or even deleterious when performed later after the stroke onset. This aggravation could be explained by the depolarisation waves which might continue to spread ischemic damage and by the speeding up of the apoptotic process leading to cell death. The effect of reperfusion on cell death through these two phenomena needs to be further studied in order to develop new therapeutic strategies for stroke patients.     

Multifaceted effects of oligodendroglial exosomes on neurons: impact on neuronal firing rate,signal transduction and gene regulation

Exosomes are small membranous vesicles of endocytic origin that are released by almost every cell type. They exert versatile functions in intercellular communication important for many physiological and pathological processes. Recently, exosomes attracted interest with regard to their role in cell–cell communication in the nervous system. We have shown that exosomes released from oligodendrocytes upon stimulation with the neurotransmitter glutamate are internalized by neurons and enhance the neuronal stress tolerance. Here, we demonstrate that oligodendroglial exosomes also promote neuronal survival during oxygen–glucose deprivation, a model of cerebral ischaemia. We show the transfer from oligodendrocytes to neurons of superoxide dismutase and catalase, enzymes which are known to help cells to resist oxidative stress. Additionally, we identify various effects of oligodendroglial exosomes on neuronal physiology. Electrophysiological analysis using in vitro multi-electrode arrays revealed an increased firing rate of neurons exposed to oligodendroglial exosomes. Moreover, gene expression analysis and phosphorylation arrays uncovered differentially expressed genes and altered signal transduction pathways in neurons after exosome treatment. Our study thus provides new insight into the broad spectrum of action of oligodendroglial exosomes and their effects on neuronal physiology. The exchange of extracellular vesicles between neural cells may exhibit remarkable potential to impact brain performance.     

Endogenous neurogenesis following ischaemic brain injury: Insights for therapeutic strategies

Ischaemic stroke is among the most common yet most intractable types of central nervous system (CNS) injury in the adult human population. In the acute stages of disease, neurons in the ischaemic lesion rapidly die and other neuronal populations in the ischaemic penumbra are vulnerable to secondary injury. Multiple parallel approaches are being investigated to develop neuroprotective, reparative and regenerative strategies for the treatment of stroke. Accumulating evidence indicates that cerebral ischaemia initiates an endogenous regenerative response within the adult brain that potentiates adult neurogenesis from populations of neural stem and progenitor cells. A major research focus has been to understand the cellular and molecular mechanisms that underlie the potentiation of adult neurogenesis and to appreciate how interventions designed to modulate these processes could enhance neural regeneration in the post-ischaemic brain. In this review, we highlight recent advances over the last 5 years that help unravel the cellular and molecular mechanisms that potentiate endogenous neurogenesis following cerebral ischaemia and are dissecting the functional importance of this regenerative mechanism following brain injury.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Brain Tissue Acidosis: Effects on the Extracellular Concentration of N-Acetylaspartate

Abstract: N -Acetylaspartate (NAA) is characterized by a high tissue-to-extracellular concentration ratio under normal conditions and is released from neurons during hyposmotic cell swelling. As cell volume regulation and acid-base homeostasis share common processes, we have examined by microdialysis whether the extracellular concentration of NAA is altered by various acidotic challenges. Twenty-minute perfusion of 50 m M NH₄⁺ through the microdialysis probe progressively lowered dialysate pH by 0.18, followed by a sudden, additional reduction after NH₄⁺ removal. The latter effect indicated extrusion of cellular H⁺ because it was suppressed by blockade of Na⁺/H⁺ exchange with 5-( N,N -dimethyl)amiloride (1 or 5 m M in perfusion medium). NH₄⁺ increased dialysate levels of NAA and lactate by approximately two- and threefold their initial values, respectively. These data demonstrate that pronounced intracellular acidosis is associated with NAA efflux, presumably from neurons. Whether this effect is linked directly to acid-base homeostasis or is secondary to acidosis-induced cell swelling remains to be clarified. Hypercapnia and perfusion of acid medium failed to increase dialysate NAA, probably because acidosis was not severe enough or the associated cellular swelling was not followed by regulatory volume decrease. As cellular swelling and acidosis are key features of cerebral ischaemia, further investigations into the role of NAA, and the development of sophisticated magnetic resonance spectroscopic methods capable of resolving intra-/extracellular NAA redistribution, would be especially relevant to clinical practice.     

Neurotransmitter synthesis in poststroke cortical neurogenesis in adult rats

Neurogenesis occurs in the cerebral cortex of adult rats after focal cerebral ischemia. Whether or not the newborn neurons could synthesize neurotransmitters is unknown. To elucidate such a possibility, a photothrombotic ring stroke model with spontaneous reperfusion was induced in adult male Wistar rats. The DNA duplication marker BrdU was repeatedly injected, and the rats were sacrificed at various times after stroke. To detect BrdU nuclear incorporation and various neurotransmitters, brain sections were processed for single/double immunocytochemistry and single/double/triple immunofluorescence. Stereological cell counting was performed to assess the final cell populations. At 48 h, 5 days, 7 days, 30 days, 60 days and 90 days after stroke, numerous cells were BrdU-immunolabeled in the penumbral cortex. Some of these were doubly immunopositive to the cholinergic neuron-specific marker ChAT or GABAergic neuron-specific marker GAD. As analyzed by 3-D confocal microscopy, the neurotransmitters acetylcholine and GABA were colocalized with BrdU in the same cortical cells. In addition, GABA was colocalized with the neuron-specific marker Neu N in the BrdU triple-immunolabeled cortical cells. This study suggests that the newborn neurons are capable of synthesizing the neurotransmitters acetylcholine and GABA in the penumbral cortex, which is one of the fundamental requisites for these neurons to function in the poststroke recovery.     

A Continuum Neuronal Model for the Instigation and Propagation of Cortical Spreading Depression

Cortical spreading depression (CSD) waves can occur in the cortices of various brain structures and are associated with the spread of depression of the electroencephalogram signal. In this paper, we present a continuum neuronal model for the instigation and spreading of CSD. Our model assumes that the brain-cell microenvironment can be treated as a porous medium consisting of extra- and intracellular compartments. The main mechanisms in our model for the transport of ions into and out of neurons are cross-membrane ionic currents and (active) pumps, coupled with diffusion in the extracellular space. To demonstrate the applicability of our model, we have carried out extensive numerical simulations under different initial conditions and inclusion of various mechanisms. Our results show that CSD waves can be instigated by injecting cross-membrane ionic currents or by applying KCl in the extracellular space. Furthermore, the estimated speeds of CSD waves are within the experimentally observed range. Effects of specific ion channels, background ion concentrations, extracellular volume fractions, and cell swelling on the propagation speed of CSD are also investigated.     

Exogenous recombinant Hsp70 mediates neuroprotection after photothrombotic stroke

Ischaemic stroke is an acute interruption of the blood supply to the brain, which leads to rapid irreversible damage to nerve tissue. Ischaemic stroke is accompanied by the development of neuroinflammation and neurodegeneration observed around the affected brain area. Heat shock protein 70 (Hsp70) facilitates cell survival under a variety of different stress conditions. Hsp70 may be secreted from cells and exhibits cytoprotective activity. This activity most likely occurs by decreasing the levels of several proinflammatory cytokines through interaction with a few receptors specific to the innate immune system. Herein, we demonstrated that intranasal administration of recombinant human Hsp70 shows a significant twofold decrease in the volume of local ischaemia induced by photothrombosis in the mouse prefrontal brain cortex. Our results revealed that intranasal injections of recombinant Hsp70 decreased the apoptosis level in the ischaemic penumbra, stimulated axonogenesis and increased the number of neurons producing synaptophysin. Similarly, in the isolated crayfish stretch receptor, consisting of a single sensory neuron surrounded by the glial envelope, exogenous Hsp70 significantly decreased photoinduced apoptosis and necrosis of glial cells. The obtained data enable one to consider human recombinant Hsp70 as a promising compound that could be translated from the bench into clinical therapies.     

Effects of nonsteroidal anti-inflammatory drugs on the uptake of various cations by lymphoid cells.

Several acidic nonsteroidal anti-inflammatory drugs (NSAID) as well as their corresponding alcohol molecules which are known to induce swelling of isolated lymphocytes by changing cell membrane permeability to water, are demonstrated also to induce changes of membrane permeability of lymphoid cells to one divalent cation, calcium, and to three monovalent cations, rubidium, cesium and sodium. According to the cells ionic environment, they increase or decrease the cellular uptake of cation which is itself also closely dependent on the ionic composition of the incubation medium. This drug-effect is very rapid, directly related to the medium NSAID concentration and almost totally reversible except to the most potent drugs such as flufenamic acid. Changes in intracellular ionic balance could have important catalytic effects on the metabolism of normal as well as of pathological cells. This fact could explain side-effects of these drugs as well as some of their therapeutic effects.     

Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

Hypoosmotic cell swelling as a novel mechanism for modulation of cloned HCN2 channels

This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to -100 mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50 nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.