Loss Of Ion Homeostasis Research Articles

328 Induced Spreading Depolarizations Produce Exaggerated Electrophysiologic Pathology in a Rat Model of Traumatic Brain Injury

INTRODUCTION: Spreading depolarizations (SDs) are waves of cortical depolarization that cause loss of ion homeostasis, brain silencing, and are prognostic of worse functional outcome in traumatic brain injury (TBI) patients. SDs occur in ∼50% of surgical TBI patients and have potential to spread from areas of injured brain into less-injured or “uninjured” cortex. Understanding differences in how injured versus uninjured cortex responds to SD waves can provide insight for therapeutic targets. METHODS: Adult Long Evans rats were subjected to either a moderate lateral fluid percussion injury (LFPI) or a sham injury. Beginning 103.4 ± 28.4 minutes after injury or sham, an electrode was placed at the site of injury (or sham) and electrocorticographic activity was recorded for two hours. SDs were induced every 15 minutes using 1M potassium chloride (KCl) soaked cotton balls placed on a frontal craniectomy separate from the recording site. Saline soaked cotton balls were used as a control. RESULTS: A total of 37 SDs were induced in the uninjured group and 32 SDs were induced in the injured group. No SDs were recorded in saline controls. There was no difference in the average time from KCl application to the start of the SD between groups (sham = 136.2, LFPI = 113.9, p = 0.4219). There was also no difference in average amplitude of the direct current (DC) shift (sham = 10.33, LFPI = 13.26, p = 0.1402). However, the injured group had longer average duration of DC shifts (sham = 53.54, LFPI = 99.22, p < 0.0001), longer average depression durations (sham = 206.6, LFPI = 265.8, p = 0.001), and larger average depth of depression (sham = 0.6824, LFPI = 0.8399, p = 0.0018). CONCLUSION: Our study revealed electrophysiologic differences between SDs induced in injured versus uninjured animals. Further studies are needed to determine the impact of these differences on neuronal damage and functional outcome.

Spinal cord injury-induced cognitive impairment: a narrative review

Spinal cord injury is a serious damage to the spinal cord that can lead to life-long disability. Based on its etiology, spinal cord injury can be classified as traumatic or non-traumatic spinal cord injury. Furthermore, the pathology of spinal cord injury can be divided into two phases, a primary injury phase, and a secondary injury phase. The primary spinal cord injury phase involves the initial mechanical injury in which the physical force of impact is directly imparted to the spinal cord, disrupting blood vessels, axons, and neural cell membranes. After the primary injury, a cascade of secondary events begins, expanding the zone of neural tissue damage, and exacerbating neurological deficits. Secondary injury is a progressive condition characterized by pro-inflammatory cytokines, reactive oxygen species, oxidative damage, excitatory amino acids such as glutamate, loss of ionic homeostasis, mitochondrial dysfunction, and cell death. This secondary phase lasts for several weeks or months and can be further subdivided into acute, subacute, and chronic. One of the most frequent and devastating complications developed among the spinal cord injury population is cognitive impairment. The risk of cognitive decline after spinal cord injury has been reported to be 13 times higher than in healthy individuals. The exact etiology of this neurological complication remains unclear, however, many factors have been proposed as potential contributors to the development of this disorder, such as concomitant traumatic brain injury, hypoxia, anoxia, autonomic dysfunction, sleep disorders such as obstructive sleep apnea, body temperature dysregulation, alcohol abuse, and certain drugs. This review focuses on a deep understanding of the pathophysiology of spinal cord injury and its relationship to cognitive impairment. We highlight the main mechanisms that lead to the development of this neurological complication in patients with spinal cord injury.

Multimodality monitoring in neurocritical care

Monitoring the health of an injured brain is essential to forewarn neurological worsening and to gain insight into the pathophysiology of a complex disorder.Clinical examination remains a cornerstone in monitoring patients with brain injury. The Glasgow coma score (GCS) is widely used but lacks information regarding brain stem functions like pupillary reaction and shows moderate inter-observer reliability. However, despite these shortcomings, GCS remains a robust indicator of the need for surgery and prognosis after cardiac arrest, hypoxic brain injury, and posterior circulation stroke. A new scoring system called Full Outline of Unresponsiveness has been proposed and shows excellent inter-observer reliability and includes points concerning brain stem functions.1 The most commonly used monitoring modality is intracranial pressure (ICP) monitoring. ICP shows threshold physiology where the outcome of the patients changes after a threshold of 20 to 25 mmHg. Refractory ICP is a good predictor of mortality but not of the functional outcome after traumatic brain injury.Brain injury causes varying degrees of disruption to cerebral blood flow and its autoregulation. Studying autoregulation provides a useful strategy for targeting cerebral perfusion pressure close to the autoregulatory range. Transcranial Doppler and ICMplus (Intensive Care Monitoring) are used to study autoregulation. ICMplus is a software-based tool that studies the correlation between slow changes in mean arterial pressure and ICP to evaluate the state of autoregulation throughout the duration of ICP monitoring.2 Brain tissue oxygen measures the partial pressure of oxygen in the extracellular fluid of the neural tissue. Reduction in brain tissue oxygen is a marker of cellular distress. A phase 2 trial on brain tissue oxygen monitoring demonstrated the safety and feasibility of the protocol-based management of brain tissue oxygenation and ICP, and a trend towards lower mortality and improved functional outcome in patients treated with combined oxygen and ICP protocol.3 Microdialysis is a point of care test that monitors substrate delivery and metabolism at the cellular level. The lactate-pyruvate (LP) ratio is an indicator of the redox state of cells and a high LP ratio is associated with an unfavourable outcome.4 The electroencephalogram (EEG) is used in critical care for monitoring sedation and diagnosis of seizure activity. EEG is a complex signal which requires advanced training and skills for interpretation. Novel EEG-based monitors are aimed at simplifying the signal for straightforward interpretation by bedside medical professionals. Cerebral function monitor (CFM) is a compressed single channel amplitude integrated EEG monitor mainly used for the detection of status epilepticus and burst suppression during thiopentone infusion. A novel technique that uses direct electrodes applied on the cortical surface called Electrocorticogram (ECoG) shows spreading depolarisations on the cortical surface that are caused by loss of ionic homeostasis and substrate delivery.5 These depolarisations are a sensitive indicator of impending neuronal death and may serve as a target for novel mechanistically oriented therapies.Detection, prevention, and monitoring of secondary cerebral insults that alter the prognosis from the injury, remains at the centre stage in neurocritical care. In the future, integrated informatics derived from multimodality monitoring will play a pivotal role in clinical decision algorithms.

Translational Advances in the Management of Acute Spinal Cord Injury

Translational Advances in the Management of Acute Spinal Cord Injury

Cold mortality is not caused by oxygen limitation or loss of ion homeostasis in the tropical freshwater shrimp Macrobrachium rosenbergii

Using the tropical crustacean Macrobrachium rosenbergii we investigate two popular hypotheses proposed to explain loss of function in ectotherms exposed to critically high and low temperatures. Specifically, we examine whether acute cold stress disrupts hemolymph and muscle ion balance or causes a loss of oxygen availability. We found that acute cold stress causes loss of righting response at 13 °C, but that a cold-induced loss of ion-balance only occurs after onset of mortality. In regards to oxygen availability, we found no decrease in hemolymph oxygen content during cold exposure, and no changes in the concentrations of the anaerobic end products l-lactate and succinate in the tail muscle of the shrimp. Therefore, our results support neither of these two popular hypotheses and it remains unknown what physiological perturbations determine the lower limits of thermal tolerance in Macrobrachium rosenbergii.

Paralysis and heart failure precede ion balance disruption in heat-stressed European green crabs

Acute exposure of ectotherms to critically high temperatures causes injury and death, and this mortality has been associated with a number of physiological perturbations including impaired oxygen transport, loss of ion and water homeostasis, and neuronal failure. It is difficult to discern which of these factors, if any, is the proximate cause of heat injury because, for example, loss of ion homeostasis can impair neuromuscular function (including cardiac function), and conversely impaired oxygen transport reduces ATP supply and can thus reduce ion transport capacity. In this study we investigated if heat stress causes a loss of ion homeostasis in marine crabs and examined if such loss is related to heart failure. We held crabs (Carcinus maenas) at temperatures just below their critical thermal maximum and measured extracellular (hemolymph) and intracellular (muscle) ion concentrations over time. Analysis of Arrhenius plots for heart rates during heating ramps revealed a breakpoint temperature below which heart rate increased with temperature, and above which heart rate declined until complete cardiac failure. As hypothesised, heat stress reduced the Nernst equilibrium potentials of both K+ and Na+, likely causing a depolarization of the membrane potential. To examine whether this loss of ion balance was likely to cause disruption of neuromuscular function, we exposed crabs to the same temperatures, but this time measured ion concentrations at the individual-specific times of complete paralysis (from which the crabs never recovered), and at the time of cardiac failure. Loss of ion balance was observed only after both paralysis and complete heart failure had occurred; indicating that the loss of neuromuscular function is not caused by a loss of ion homeostasis. Instead we suggest that the observed loss of ion balance may be linked to tissue damage related to heat death.

Computational Analysis of AMPK-Mediated Neuroprotection Suggests Acute Excitotoxic Bioenergetics and Glucose Dynamics Are Regulated by a Minimal Set of Critical Reactions.

Loss of ionic homeostasis during excitotoxic stress depletes ATP levels and activates the AMP-activated protein kinase (AMPK), re-establishing energy production by increased expression of glucose transporters on the plasma membrane. Here, we develop a computational model to test whether this AMPK-mediated glucose import can rapidly restore ATP levels following a transient excitotoxic insult. We demonstrate that a highly compact model, comprising a minimal set of critical reactions, can closely resemble the rapid dynamics and cell-to-cell heterogeneity of ATP levels and AMPK activity, as confirmed by single-cell fluorescence microscopy in rat primary cerebellar neurons exposed to glutamate excitotoxicity. The model further correctly predicted an excitotoxicity-induced elevation of intracellular glucose, and well resembled the delayed recovery and cell-to-cell heterogeneity of experimentally measured glucose dynamics. The model also predicted necrotic bioenergetic collapse and altered calcium dynamics following more severe excitotoxic insults. In conclusion, our data suggest that a minimal set of critical reactions may determine the acute bioenergetic response to transient excitotoxicity and that an AMPK-mediated increase in intracellular glucose may be sufficient to rapidly recover ATP levels following an excitotoxic insult.

Pore-Forming Toxins Induce Macrophage Necroptosis during Acute Bacterial Pneumonia.

Necroptosis is a highly pro-inflammatory mode of cell death regulated by RIP (or RIPK)1 and RIP3 kinases and mediated by the effector MLKL. We report that diverse bacterial pathogens that produce a pore-forming toxin (PFT) induce necroptosis of macrophages and this can be blocked for protection against Serratia marcescens hemorrhagic pneumonia. Following challenge with S. marcescens, Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, uropathogenic Escherichia coli (UPEC), and purified recombinant pneumolysin, macrophages pretreated with inhibitors of RIP1, RIP3, and MLKL were protected against death. Alveolar macrophages in MLKL KO mice were also protected during S. marcescens pneumonia. Inhibition of caspases had no impact on macrophage death and caspase-1 and -3/7 were determined to be inactive following challenge despite the detection of IL-1β in supernatants. Bone marrow-derived macrophages from RIP3 KO, but not caspase-1/11 KO or caspase-3 KO mice, were resistant to PFT-induced death. We explored the mechanisms for PFT-induced necroptosis and determined that loss of ion homeostasis at the plasma membrane, mitochondrial damage, ATP depletion, and the generation of reactive oxygen species were together responsible. Treatment of mice with necrostatin-5, an inhibitor of RIP1; GW806742X, an inhibitor of MLKL; and necrostatin-5 along with co-enzyme Q10 (N5/C10), which enhances ATP production; reduced the severity of S. marcescens pneumonia in a mouse intratracheal challenge model. N5/C10 protected alveolar macrophages, reduced bacterial burden, and lessened hemorrhage in the lungs. We conclude that necroptosis is the major cell death pathway evoked by PFTs in macrophages and the necroptosis pathway can be targeted for disease intervention.

Exposure to acute severe hypoxia leads to increased urea loss and disruptions in acid-base and ionoregulatory balance in dogfish sharks (Squalus acanthias).

The effects of acute moderate (20% air O2 saturation; 6-h exposure) and severe (5% air O2 saturation; 4-h exposure) hypoxia on N-waste, acid-base, and ion balance in dogfish sharks (Squalus acanthias suckleyi) were evaluated. We predicted that the synthesis and/or retention of urea, which are active processes, would be inhibited by hypoxia. Exposure to moderate hypoxia had negligible effects on N-waste fluxes or systemic physiology, except for a modest rise in plasma lactate. Exposure to severe hypoxia led to a significant increase in urea excretion (Jurea), while plasma, liver, and muscle urea concentrations were unchanged, suggesting a loss of urea retention. Ammonia excretion (Jamm) was elevated during normoxic recovery. Moreover, severe hypoxia led to disruptions in acid-base balance, indicated by a large increase in plasma [lactate] and substantial decreases in arterial pHa and plasma [Formula: see text], as well as loss of ionic homeostasis, indicated by increases in plasma [Mg(2+)], [Ca(2+)], and [Na(+)]. We suggest that severe hypoxia in dogfish sharks leads to a reduction in active gill homeostatic processes, such as urea retention, acid-base regulation and ionoregulation, and/or an osmoregulatory compromise due to increased functional gill surface area. Overall, the results provide a comprehensive picture of the physiological responses to a severe degree of hypoxia in an ancient fish species.

PEG-PDLLA Micelle Treatment Improves Axonal Function of the Corpus Callosum following Traumatic Brain Injury

The initial pathological changes of diffuse axonal injury following traumatic brain injury (TBI) include membrane disruption and loss of ionic homeostasis, which further lead to dysfunction of axonal conduction and axon disconnection. Resealing the axolemma is therefore a potential therapeutic strategy for the early treatment of TBI. Monomethoxy poly (ethylene glycol)-poly (D, L-lactic acid) di-block copolymer micelles (mPEG-PDLLA) have been shown to restore depressed compound action potentials (CAPs) of spinal axons and promote functional recovery after spinal cord injury. Here, we evaluate the effect of the micelles on repairing the injured cortical axons following TBI. Adult mice subjected to controlled cortical impact (CCI) were treated with intravenous injection of the micelles at 0 h or 4 h after injury. Evoked CAPs were recorded from the corpus callosum of coronal cortical slices at 2 days after injury. The CCI caused significant decreases in the amplitudes of two CAP peaks that were respectively generated by the faster myelinated axons and slower unmyelinated axons. Micelle treatment at both 0 h and 4 h after CCI resulted in significant increases in both CAP peak amplitudes. Injection of fluorescent dye-labeled micelles revealed high fluorescent staining in cortical gray and white matters underneath the impact site. Labeling membrane-perforated neurons by injecting a membrane impermeable dye Texas Red-labeled dextran into lateral ventricles at 2 h post-CCI revealed that immediate micelle injection after CCI did not reduce the number of dye-stained cortical neurons and dentate granule cells of the hippocampus, indicating its ineffectiveness in repairing plasma membrane of neuronal somata. We conclude that intravenous administration of mPEG-PDLLA micelles immediately or at 4 h after TBI allows brain penetration via the compromised blood brain-barrier, and thereby improves the function of both myelinated and unmyelinated axons of the corpus callosum.

Why do insects enter and recover from chill coma? Low temperature and high extracellular potassium compromise muscle function in Locusta migratoria.

When exposed to low temperatures, many insect species enter a reversible comatose state (chill coma), which is driven by a failure of neuromuscular function. Chill coma and chill coma recovery have been associated with a loss and recovery of ion homeostasis (particularly extracellular [K(+)], [K(+)]o) and accordingly onset of chill coma has been hypothesized to result from depolarization of membrane potential caused by loss of ion homeostasis. Here, we examined whether onset of chill coma is associated with a disturbance in ion balance by examining the correlation between disruption of ion homeostasis and onset of chill coma in locusts exposed to cold at varying rates of cooling. Chill coma onset temperature changed maximally 1°C under different cooling rates and marked disturbances of ion homeostasis were not observed at any of the cooling rates. In a second set of experiments, we used isolated tibial muscle to determine how temperature and [K(+)]o, independently and together, affect tetanic force production. Tetanic force decreased by 80% when temperature was reduced from 23°C to 0.5°C, while an increase in [K(+)]o from 10 mmol l(-1) to 30 mmol l(-1) at 23°C caused a 40% reduction in force. Combining these two stressors almost abolished force production. Thus, low temperature alone may be responsible for chill coma entry, rather than a disruption of extracellular K(+) homeostasis. As [K(+)] also has a large effect on tetanic force production, it is hypothesized that recovery of [K(+)]o following chill coma could be important for the time to recovery of normal neuromuscular function.

Mitochondrial Permeability Transition as Target of Anticancer Drugs

Mitochondria are the cell powerhouses but also contain the mechanisms leading to cell death. Many signals converge on mitochondria to cause the permeabilization of mitochondrial membranes by the mitochondrial permeability transition (MPT) induction and the opening of transition pores (PTPs). These events cause loss of ionic homeostasis, matrix swelling, outer membrane rupture leading to pro-apoptotic factors release, and impairment of bioenergetics functions. The molecular mechanism underlying MPT induction is not completely elucidated however, a growing body of evidence supports the concept that pharmacological induction of PTPs in mitochondria of neoplastic cells is an effective and promising strategy for therapeutic approaches against cancer. The first part of this article presented as a review also evidences the main constituents of PTP and several compounds targeting them for inducing the phenomenon. The second part of the article regards the recent experimental development in the field, in particular, the effects of peniocerol (PEN), a sterol isolated from the root of Myrtillocactus geometrizans, at cellular and mitochondrial level. PEN exhibits a cytotoxic activity on some human tumor cell lines, whose mechanism is attributable to the oxidation of critical thiols located on adenine nucleotide translocase, the protein mainly involved in PTP. This event in the presence of Ca(2+) induces the MPT with the release of the pro-apoptotic factors cytochrome c and apoptosis inducing factor. These observations evidence that PEN may trigger both the caspase-dependent and caspaseindependent apoptotic pathways. This characteristic renders PEN a very interesting compound that could be developed to obtain more effective antiproliferative agents targeting mitochondria for anticancer therapy.

Na-K-Cl Cotransporter-1 as a Regulator of Manganese-induced Astrocyte Swelling

Astrocyte swelling leads to brain edema, intracranial pressure, brain herniation and acute liver failure (fulminant hepatic failure) which is the major cause of death in this condition. Manganese has been strongly implicated as an important factor in astrocyte swelling. Manganese in excess is neurotoxic and causes a CNS disorder that resembles Parkinson¡¦s disease (manganism). Manganese highly accumulates in astrocytes, which renders these cells more vulnerable to its toxicity. In addition to manganism, increased brain levels of manganese have been found in hepatic encephalopathy. Manganese is known to cause cellswelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter-1 (NKCC1) has been shown to be involved in cell swelling in several neurological disorders.We therefore examined the effect of manganese on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to manganese (50 µM), and NKCC activity was measured. Manganese increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished manganese-induced astrocyte swelling. Manganese (Mn) also increased total as well as phosphorylated NKCC1. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by manganese and that such activation appears to be mediated by NKCC1 abundance.

Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited.

Carbimazole Is an Inhibitor of Protein Synthesis and Protects from Neuronal Hypoxic Damage In Vitro

Oxygen deprivation during ischemic or hemorrhagic stroke results in ATP depletion, loss of ion homeostasis, membrane depolarization, and excitotoxicity. Pharmacologic restoration of cellular energy supply may offer a promising concept to reduce hypoxic cell injury. In this study, we investigated whether carbimazole, a thionamide used to treat hyperthyroidism, reduces neuronal cell damage in oxygen-deprived human SK-N-SH cells or primary cortical neurons. Our results revealed that carbimazole induces an inhibitory phosphorylation of eukaryotic elongation factor 2 (eEF2) that was associated with a marked inhibition of global protein synthesis. Translational inhibition resulted in significant bioenergetic savings, preserving intracellular ATP content in oxygen-deprived neuronal cells and diminishing hypoxic cellular damage. Phosphorylation of eEF2 was mediated by AMP-activated protein kinase and eEF2 kinase. Carbimazole also induced a moderate calcium influx and a transient cAMP increase. To test whether translational inhibition generally diminishes hypoxic cell damage when ATP availability is limiting, the translational repressors cycloheximide and anisomycin were used. Cycloheximide and anisomycin also preserved ATP content in hypoxic SK-N-SH cells and significantly reduced hypoxic neuronal cell damage. Taken together, these data support a causal relation between the pharmacologic inhibition of global protein synthesis and efficient protection of neurons from ischemic damage by preservation of high-energy metabolites in oxygen-deprived cells. Furthermore, our results indicate that carbimazole or other translational inhibitors may be interesting candidates for the development of new organ-protective compounds. Their chemical structure may be used for computer-assisted drug design or screening of compounds to find new agents with the potential to diminish neuronal damage under ATP-limited conditions.

The Potential of Tetrandrine as a Protective Agent for Ischemic Stroke

Stroke is one of the leading causes of mortality, with a high incidence of severe morbidity in survivors. The treatment to minimize tissue injury after stroke is still unsatisfactory and it is mandatory to develop effective treatment strategies for stroke. The pathophysiology of ischemic stroke is complex and involves many processes including energy failure, loss of ion homeostasis, increased intracellular calcium level, platelet aggregation, production of reactive oxygen species, disruption of blood brain barrier, and inflammation and leukocyte infiltration, etc. Tetrandrine, a bisbenzylisoquinoline alkaloid, has many pharmacologic effects including anti-inflammatory and cytoprotective effects. In addition, tetrandrine has been found to protect the liver, heart, small bowel and brain from ischemia/reperfusion injury. It is a calcium channel blocker, and can inhibit lipid peroxidation, reduce generation of reactive oxygen species, suppress the production of cytokines and inflammatory mediators, inhibit neutrophil recruitment and platelet aggregation, which are all devastating factors during ischemia/reperfusion injury of the brain. Because tetrandrine can counteract these important pathophysiological processes of ischemic stroke, it has the potential to be a protective agent for ischemic stroke.

The Na–K–Cl Co-transporter in astrocyte swelling

Ion channels, exchangers and transporters are known to be involved in cell volume regulation. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na(+)-K(+)-Cl(-) cotransporters has been shown to regulate cell volume in many conditions. The Na(+)-K(+)-Cl- cotransporters (NKCC) are a class of membrane proteins that transport Na, K, and Cl ions into and out of a wide variety of epithelial and nonepithelial cells. Studies have established the role of NKCC1 in astrocyte swelling/brain edema in ischemia and trauma. Our recent studies suggest that NKCC1 activation is also involved in astrocyte swelling induced by ammonia and in the brain edema in the thioacetamide model of acute liver failure. This review will focus on mechanisms of NKCC1 activation and its contribution to astrocyte swelling/brain edema in neurological disorders, with particular emphasis on ammonia neurotoxicity and acute liver failure.

Na-K-Cl Cotransporter-1 in the Mechanism of Ammonia-induced Astrocyte Swelling

Brain edema and the consequent increase in intracranial pressure and brain herniation are major complications of acute liver failure (fulminant hepatic failure) and a major cause of death in this condition. Ammonia has been strongly implicated as an important factor, and astrocyte swelling appears to be primarily responsible for the edema. Ammonia is known to cause cell swelling in cultured astrocytes, although the means by which this occurs has not been fully elucidated. A disturbance in one or more of these systems may result in loss of ion homeostasis and cell swelling. In particular, activation of the Na-K-Cl cotransporter (NKCC1) has been shown to be involved in cell swelling in several neurological disorders. We therefore examined the effect of ammonia on NKCC activity and its potential role in the swelling of astrocytes. Cultured astrocytes were exposed to ammonia (NH(4)Cl; 5 mm), and NKCC activity was measured. Ammonia increased NKCC activity at 24 h. Inhibition of this activity by bumetanide diminished ammonia-induced astrocyte swelling. Ammonia also increased total as well as phosphorylated NKCC1. Treatment with cyclohexamide, a potent inhibitor of protein synthesis, diminished NKCC1 protein expression and NKCC activity. Since ammonia is known to induce oxidative/nitrosative stress, and antioxidants and nitric-oxide synthase inhibition diminish astrocyte swelling, we also examined whether ammonia caused oxidation and/or nitration of NKCC1. Cultures exposed to ammonia increased the state of oxidation and nitration of NKCC1, whereas the antioxidants N-nitro-l-arginine methyl ester and uric acid all significantly diminished NKCC activity. These agents also reduced phosphorylated NKCC1 expression. These results suggest that activation of NKCC1 is an important factor in the mediation of astrocyte swelling by ammonia and that such activation appears to be mediated by NKCC1 abundance as well as by its oxidation/nitration and phosphorylation.

Modulation of CNS function by natural extracts

The “amyloid cascade hypothesis” predicts that the progress for pathogenesis of Alzheimer's disease (AD) involves multiple factors such as amyloid aggregation, oxidative stress, loss of ion homeostasis and inflammation. In a transgenic C. elegans model of AD, intracellular expression of human Aβ is associated with elevated oxidative stress, accumulation of Ab aggregation and progressive paralysis behavior. Recently we demonstrated that EGb 761 stimulated neurogenesis in aged wild type mice as well as a mouse model of Alzheimer's disease, and altered neurotransmitter serotonin (5-HT) sensitivity in a transgenic Aβ-expressing C. elegans. Serotonin plays an important role in neuronal plasticity and survival. It has also been associated with behavioral deficiencies seen in AD patients. The aim of this study is to determine the mechanism of action of EGb 761 and its constituents on 5-HT transmission. Several C. elegans 5-HT system mutants were used for 5-HT-controlled behavioral test. Our results show that wild type worms treated with EGb 761 demonstrate a similar sensitivity to 5-HT as the untreated mod-1 mutants, suggesting that the effect of EGb 761 on locomotion depends on the postsynaptic 5-HT receptors. Bilobalide also had a significant inhibitory effect on the egg-laying response in the wild type animals and this effect was not blocked in mod-5 mutant worms, further confirming postsynaptic action. Moreover, combinations of compounds with different mechanisms of action exhibit potential synergistic and/or additive effects. These results may provide a rationale for more effective natural therapeutic strategies for the prevention and/or treatment of AD.

Calcium, the Universe and Everything

AbstractCalcium plays a pivotal role in lens physiology and pathophysiology. I will review research from the Norwich Eye Lab, which has been fundamental in establishing calcium and its function in the lens.Human cortical cataract is associated with a loss of ionic homeostasis, with calcium overload a hallmark of this disease. Free calcium has also been found to increase in non‐cataractous human lenses as a result of aging, suggesting that it may be an early event in cataractogenesis. Lens organ culture experiments, using bovine and human lenses, have shown that calcium overload is indeed fundamental in loss of lens transparency. Raised intracellular calcium initiated proteolytic events both in the soluble (crystallin) fraction and the cytoskeletal fraction, supporting a role for the calcium‐activated protease calpain in the opacification process. In order to understand mechanisms of calcium overload, it is also important to understand the mechanisms of normal calcium homeostasis in the lens. This has been extensively studied, mainly in lens epithelial cells, with a particular emphasis on the role of the endoplasmic reticulum calcium store. Investigation of calcium signaling has, in addition to this, shown the lens to have a remarkable and diverse pharmacology, with functional expression of a plethora of receptors which signal via calcium including muscarinic receptors, purinoceptors and EGF receptors. Recent work in the lab has also extended this interest in calcium in the lens to other ocular cells types, where calcium signaling could have a role to play in other eye diseases such as retinal detachment and glaucoma.Investigation of calcium in the lens has demonstrated a central role in the pathophysiology of cortical cataract. By understanding the molecular mechanisms underlying cataractogenesis we move closer to the development of pharmacological strategies for the treatment of this disease.