Release Of Excitotoxic Amino Acids Research Articles

Roles of Nitric Oxide in Brain Ischemia and Reperfusion.

Brain ischemia and reperfusion (I/R) is one of the most severe clinical manifestations of ischemic stroke, placing a significant burden on both individuals and society. The only FDA-approved clinical treatment for ischemic stroke is tissue plasminogen activator (t-PA), which rapidly restores cerebral blood flow but can have severe side effects. The complex pathological process of brain I/R has been well-established in the past few years, including energy metabolism disorders, cellular acidosis, doubling of the synthesis or release of excitotoxic amino acids, intracellular calcium homeostasis, free radical production, and activation of apoptotic genes. Recently, accumulating evidence has shown that NO may be strongly related to brain I/R and involved in complex pathological processes. This review focuses on the role of endogenous NO in pathological processes in brain I/R, including neuronal cell death and blood brain barrier disruption, to explore how NO impacts specific signaling cascades and contributes to brain I/R injury. Moreover, NO can rapidly react with superoxide to produce peroxynitrite, which may also mediate brain I/R injury, which is discussed here. Finally, we reveal several therapeutic approaches strongly associated with NO and discuss their potential as a clinical treatment for ischemic stroke.

The Seek of Neuroprotection: Introducing Cannabinoids

The cannabinoid system is constituted by some endogenous ligands (endocannabinoids), usually arachydonic acid derivatives, and their specific receptors. The endogenous cannabinoid system (ECS) is involved in the control of synaptic transmission, modulating memory, motivation, movement, nociception, appetite and thermoregulation. ECS also exert extraneural effects, mainly immunomodulation and vasodilation. Two cannabinoid receptors have been cloned so far: CB(1) receptors are expressed in the central nervous system (CNS) but can also be found in glial cells and in peripheral tissues; CB(1) receptors are Gi/o protein coupled receptors that modulate the activity of several plasma membrane proteins and intracellular signaling pathways. CB(2) receptors are also Gi/o protein-coupled receptors; although it is accepted that CB(2) receptors are not expressed in forebrain neurons, they have been described in activated glia. Some of the cannabinoids activate other receptors, for instance vanilloid receptors (TRPV1). Lately, the ECS is emerging as a natural system of neuroprotection. This consideration is based on some properties of cannabinoids as their vasodilatory effect, the inhibition of the release of excitotoxic amino acids and cytokines, and the modulation of oxidative stress and toxic production of nitric oxide. Such effects have been demonstrated in adult and newborn animal models of acute and chronic neurodegenerative conditions, and postulate cannabinoids as valuable neuroprotective agents. Patents related to cannabinoid receptors are also discussed.

Localized therapeutic hypothermia in the brain for the treatment of ischemic stroke

stroke is, according to the World Health Organization ([10][1]), the third most common cause of death in the developed countries, exceeded only by coronary heart disease and cancer. Annually, 15 million people worldwide suffer a stroke. Of these 15 million people, 5.5 million die and 5 million are

El sistema cannabinoide y su importancia en el período perinatal

The cannabinoid system has been recently described, including the endogenous ligands, mainly arachidonic acid derivatives, and their specific receptors. Endocannabinoids are involved in the modulation of synaptic transmission, through which they exert their psychoactive, motor and antinociceptive effects, among others; they also exert extraneural effects, mainly immunomodulation and vasodilation. Recent data suggest that the cannabinoid system might play an important role in human ontogeny and could participate in the implantation and early development of the embryo, in fetal brain development, and in the beginning of breast feeding after birth. In addition, the vasodilatory effect of cannabinoids, together with inhibition of the release of excitotoxic amino acids and cytokines, as well as modulation of oxidative stress and the toxic production of nitric oxide, justify the growing evidence pointing to a possible neuroprotective effect of cannabinoids in perinatal asphyxia.

Increased allopregnanolone levels in the fetal sheep brain following umbilical cord occlusion.

Allopregnanolone (AP) is a potent modulator of the GABAA receptor. Brain AP concentrations increase in response to stress, which is thought to provide neuroprotection by reducing excitation in the adult brain. Umbilical cord occlusion (UCO) causes hypoxia and asphyxia in the fetus, which are major risk factors associated with poor neurological outcome for the neonate, and may lead to adverse sequelae such as cerebral palsy. The aims of this study were as follows: (i) to determine the effect of 10 min UCO on AP concentrations in the extracellular fluid of the fetal brain using microdialysis, and (ii) to compare the content of the steroidogenic enzymes P450scc and 5alpha-reductase type II (5alphaRII) with brain and CSF neurosteroid concentrations. UCO caused fetal asphyxia, hypertension, bradycardia and respiratory acidosis, which returned to normal levels after 1-2 h. AP concentrations in dialysate samples from probes implanted in grey and white matter of the parietal cortex were significantly increased 1 h after UCO from control levels of 10.4 +/- 0.4 and 12.4 +/- 0.3 to 26.0 +/- 5.1 and 27.6 +/- 6.4 nmol l(-1), respectively (P < 0.05), before returning to pre-occlusion levels by 3-4 h after UCO. When fetal brains were collected 1 h after a 10 min UCO, the relative increases of AP and pregnenolone content in the parietal cortex were similar to the increase observed in the extracellular (dialysate) fluid. AP, but not pregnenolone, was increased in CSF at this time. P450scc and 5alphaRII enzyme expression was significantly increased in the cerebral cortex in the UCO fetuses compared to control fetuses. These results suggest that the fetal brain is capable of transiently increasing neurosteroid production in response to asphyxia. The action of the increased neurosteroid content at GABAA receptors may serve to diminish the increased excitation due to excitotoxic amino acid release, and provide short-term protection to brain cells during such stress.

RCBF in hemorrhagic, non-hemorrhagic and mixed contusions after severe head injury and its effect on perilesional cerebral blood flow.

Intracerebral contusions can lead to regional ischemia caused by extensive release of excitotoxic aminoacids leading to increased cytotoxic brain edema and raised intracranial pressure. rCBF measurements might provide further information about the risk of ischemia within and around contusions. Therefore, the aim of the presented study was to compare the intra- and perilesional rCBF of hemorrhagic, non-hemorrhagic and mixed intracerebral contusions. In 44 patients, 60 stable Xenon-enhanced CT CBF-studies were performed (EtCO2 30 +/- 4 mmHg SD), initially 29 hours (39 studies) and subsequent 95 hours after injury (21 studies). All lesions were classified according to localization and lesion type using CT/MRI scans. The rCBF was calculated within and 1-cm adjacent to each lesion in CT-isodens brain. The rCBF within all contusions (n = 100) of 29 +/- 11 ml/100 g/min was significantly lower (p < 0.0001, Mann-Whitney U) compared to perilesional rCBF of 44 +/- 12 ml/100 g/min and intra/perilesional correlation was 0.4 (p < 0.0005). Hemorrhagic contusions showed an intra/perilesional rCBF of 31 +/- 11/44 +/- 13 ml/100 g/min (p < 0.005), non-hemorrhagic contusions 35 +/- 13/46 +/- 10 ml/100 g/min (p < 0.01). rCBF in mixed contusions (25 +/- 9/44 +/- 12 ml/100 g/min, p < 0.0001) was significantly lower compared to hemorrhagic and non-hemorrhagic contusions (p < 0.02). Intracontusional rCBF is significantly reduced to 29 +/- 11 ml/100 g/min but reduced below ischemic levels of 18 ml/100 g/min in only 16% of all contusions. Perilesional CBF in CT normal appearing brain closed to contusions is not critically reduced. Further differentiation of contusions demonstrates significantly lower rCBF in mixed contusions (defined by both hyper- and hypodense areas in the CT-scan) compared to hemorrhagic and non-hemorrhagic contusions. Mixed contusions may evolve from hemorrhagic contusions with secondary increased perilesional cytotoxic brain edema leading to reduced cerebral blood flow and altered brain metabolism. Therefore, the treatment of ICP might be individually modified by the measurement of intra- and pericontusional cerebral blood.

Hyposmotically induced amino acid release from the rat cerebral cortex: role of phospholipases and protein kinases

In an evaluation of the contribution of swelling-induced amino acid release, through the regulatory volume decrease (RVD) process, to cerebral ischemic injury, studies of the role of phospholipases and protein kinases in the response to hyposmotic stress were undertaken using an in vivo rat cortical cup model. Hyposmotic stress induced significant releases of aspartate, glutamate, glycine, phosphoethanolamine, taurine and GABA from the rat cerebral cortex. Taurine release was most affected, exhibiting a greater than 9-fold increase during the hyposmotic stimulus. The phospholipase A 2 (PLA 2) inhibitors 4-bromophenacyl bromide (1 μM) and 7,7-dimethyleicosadienoic acid (5 μM) had no significant effects on hyposmotically induced amino acid release. AACOCF 3 (50 μM), an inhibitor of cytosolic PLA 2 decreased taurine release to 84% of DMSO controls. The release of the other amino acids was not affected. The phospholipase C inhibitor U73122 (5 μM) had no significant effects on amino acid release. The protein kinase C (PKC) inhibitor chelerythrine (5 μM) significantly reduced hyposmotically induced taurine release to 72% of saline controls but had no significant effects on the other amino acids. Stimulation of PKC with phorbol 12-myristate, 13-acetate (10 μM) did not significantly change taurine, glutamate, glycine or phosphethanolamine release. The releases of aspartate and GABA were enhanced 2 to 3 fold. Phorbol 12,13-didecanoate (10 μM), another potent stimulator of PKC, significantly increased taurine release to 122% of DMSO controls. The releases of aspartate, glutamate and glycine were enhanced 2.5 to 3.5 fold. Similarly, stimulation of protein kinase A with forskolin (100 μM) significantly increased taurine, aspartate, and glycine release 1.5- to 2-fold compared to DMSO controls. In summary, phospholipases may play a minor role in volume regulation. These studies also support the hypothesis that protein kinases play a modulatory role in the RVD response. The results show that although RVD may play a role, additional mechanisms, including phospholipase activation, must be involved in the ischemia-evoked release of excitotoxic amino acids.

Attenuated Neurotransmitter Release and Spreading Depression-Like Depolarizations after Focal Ischemia in Mutant Mice with Disrupted Type I Nitric Oxide Synthase Gene

Nitric oxide (NO) plays a complex role in the pathophysiology of cerebral ischemia. In this study, mutant mice with disrupted type I (neuronal) NO synthase (nNOS) were compared with wild-type littermates after permanent focal ischemia. Cerebral blood flow in the central and peripheral zones of the ischemic distribution were measured with laser doppler flowmetry. Simultaneously, microdialysis electrodes were used to measure extracellular amino acid concentrations and DC potential in these same locations. Blood flow was reduced to <25 and 60% of baseline levels in the central and peripheral zones, respectively; there were no differences in nNOS mutants versus wild-type mice. Within the central ischemic zone, DC potentials rapidly shifted to -20 mV in all mice. In the ischemic periphery, spreading depression (SD)-like waves of depolarization were observed. SD-like events were significantly fewer in the nNOS mutant mice. Concurrent with these hemodynamic and electrophysiological perturbations, extracellular elevations in amino acids occurred after ischemia. There were no detectable differences between wild-type and mutant mice in the ischemic periphery. However, in the central zone of ischemia, elevations in glutamate and GABA were significantly lower in the nNOS mutants. Twenty-four hour infarct volumes in the nNOS mutant mice were significantly smaller than in their wild-type littermates. Overall, the number of SD-like depolarizations and the integrated efflux of glutamate were significantly correlated with infarct size. These results suggest that NO derived from the nNOS isoform contributes to tissue damage after focal ischemia by amplifying excitotoxic amino acid release in the core and deleterious waves of SD-like depolarizations in the periphery.

Neuronal death: is there a role for astrocytes?

Astrocytes are ubiquitous in the brain and have multiple functions. It is becoming increasingly clear that they play an important role in monitoring the neuromicroenvironment in CNS and in information processing or signaling in the nervous system in normal conditions and respond to CNS injuries in a gradual and varied way. It is still debated whether such reactions are beneficial or detrimental. It was believed that reactive astrogliosis observed in most neurological disorders may regulate the removal of toxic compounds produced by damaged neurons and support neuronal growth by releasing trophic factors. However it was also suggested that astrocytes contribute to a decline of neurologic function, for example by accumulation and release of excitotoxic aminoacids after ischemia and oxidative stress, formation of epileptogenic scars in response to CNS injury and metabolism of protoxins to potent toxins. In a number of metabolic diseases astrocytes, not neurons, may be the primary target. The astrocyte's role in normal and pathological conditions will be discussed in the light of recent information about their metabolism, receptor distribution and release.

The significance of glucose turnover in the brain in the pathogenetic mechanisms of Alzheimer's disease.

This paper presents a comprehensive survey of the pathogenesis and pathophysiology of Alzheimer's disease (AD). Two mechanisms are of etiological importance in the development of a degenerative dementing brain disease: 1. Lesions in the mitochondrial genome that are caused by free radicals. Primary degenerative AD is characterized by a tendency to acquire random lesions within mitochondrial DNA that are produced by free radicals. The consequence of these lesions is a decrease in glucose turnover and a decline in oxidative phosphorylation. Point mutations on chromosome 21 are hypothesized to increase the susceptibility of mitochondrial DNA to lesions created by free radicals. 2. Ischemic brain lesions as well as traumatic brain damage cause an increase in the release of excitotoxic amino acids (glutamate, aspartate, etc.). These neurotransmitters increase CA(+2) influx into the nerve cell and significantly lower energy production. From a pathogenetic point of view, AD is characterized by a decrease in glucose turnover in the brain. The progression of AD can be monitored by F18- deoxyglucose PET studies. This technique also allows the recognition of patients who are prone to develop AD. The actual development of a cognitive deficit is a threshold phenomenon that occurs if glucose turnover in the hippocampus or temporoparietal cortex drops below a critical level of about 40% of the level of age-matched controls. The low glucose turnover in AD causes a cholinergic deficit by decreasing the synthesis of AcCoA, which is used by choline acetyltransferase in the acetylation of choline to acetylcholine. The decrease in glucose turnover also reduces oxidative phosphorylation. The resulting decrease in ATP triggers the hyperphosphorylation of tau protein by activating protein kinase 40erk. The hyperphosphorylation leads to the development of paired helical filaments. The generation of beta amyloid and the loss of neuronal synapses are also caused by a decrease in oxidative phosphorylation, since beta amyloid precursor proteins are not inserted into the membranes of nerve cells in the absence of a sufficient amount of ATP. This results in the generation of intact beta amyloid molecules and leads to amyloidosis in the brains of patients with Alzheimer's disease.

Arachidonic acid and lysophosphatidylcholine modulate excitatory transmitter amino acid release from the rat cerebral cortex

The involvement of phospholipase A 2 (PLA 2) products in ischemia-evoked release of excitatory neurotransmitter amino acids (EAAs) from the cerebral cortex was studied in a four vessel occlusion rat model of cerebral ischemia/reperfusion. In comparison with untreated animals, arachidonic acid (AA; 5 × 10 −7 M to 5 × 10 −5 M) significantly reduced the ischemia-evoked efflux of glutamate and aspartate into cortical superfusates. Direct application of lysophosphatidylcholine (LysoPC; 55.4 μg/ml) to the cerebral cortex of non-ischemic animals resulted in a significant increase in glutamate levels. These results indicate that the immediate products of PLA 2 action on plasma membrane phospholipids can either enhance or inhibit excitotoxic amino acid release following cerebral ischemia. The effect of AA is likely to be a result of its ability to inhibit PLA 2; that of LysoPC, a consequence of its detergent action.

Distinct neuroprotective profiles for σ ligands against N-methyl- d-aspartate (NMDA), and hypoxia-mediated neurotoxicity in neuronal culture toxicity studies

Substantiating evidence has raised the possibility that σ ligands may have therapeutic potential as neuroprotective agents in brain ischemia. It has been suggested that the neuroprotective capacity of σ ligands is related primarily to their affinity for the NMDA receptor complex and not to any selective action at the σ binding site. However, σ specific ligands, devoid of significant affinity for the NMDA receptor, are also neuroprotective via an inhibition of the ischemic-induced presynaptic release of excitotoxic amino acids. In the present study, we have investigated the potential neuroprotective effect of a comprehensive series of σ ligands, with either significant (σ/PCP) or negligible (σ) affinity for the PCP site of the NMDA receptor, in order to delineate a selective σ site-dependent neuroprotective effect. For this aim, we have employed two different neuronal culture toxicity paradigms implicating either postsynaptic-mediated neurotoxicity, (brief exposure of cultures to a low concentration of NMDA or Kainate) or pre- and postsynaptic mechanisms (exposure to hypoxic/hypoglycemic conditions). Only σ ligands with affinity for the NMDA receptor [(+) and (−) cyclazocine, (+) pentazocine, (+) SKF-10047, ifenprodil and haloperidol] were capable of attenuating NMDA-induced toxicity whereas the σ [(+)BMY-14802, DTG, JO1784, JO1783, and (+)3-PPP] and κ-opioid [CI-977, U-50488H] ligands, with very low affinity for the NMDA receptor, were inactive. The rank order of potency, based on the 50% protective concentration (PC 50) value, of σ/PCP ligands against NMDA-mediated neurotoxicity correlates with their affinity for the PCP site of the NMDA receptor, and not with their affinity for the σ site. In addition σ/PCP, σ or κ-opioid ligands failed to attenuate kainate-mediated neurotoxicity. On the other hand, σ/PCP, σ and κ-opioid ligands were potent inhibitors of hypoxia/hypoglycemia-induced neurotoxicity, although their neuroprotective potency did not correlate with their affinity for either the σ or PCP binding sites. In conclusion, the ability of σ and κ-opioid ligands to attenuate hypoxia/hypoglycemia, but not NMDA or kainate-induced toxicity, suggests that these drugs exert their neuroprotective role by a predominantly presynaptic mechanism possibly by inhibiting ischemic-mediated glutamate release.

CGS 15943, An adenosine A 2 receptor antagonist, reduces cerebral ischemic injury in the mongolian gerbil

The adenosine A 2 receptor antagonist CGS 15943 (0.1 mg/kg, i.p.) was tested for cerebroprotective activity in a gerbil stroke model. CGS 15943 markedly reduced stroke injury assessed by locomotor activity monitoring and by histopathological measurement of hippocampal CA1 pyramidal cell injury. It is proposed that a previously demonstrated reduction in the ischemia/reperfusion-evoked release of excitotoxic amino acids following CGS 15943 administration could account for its cerebroprotective actions.

Ischemia does not induce the release of excitotoxic amino acids from the hippocampus of newborn rats

Simulated ischemic conditions or a source of oxygen-derived free radicals, such as xanthine plus xanthine oxidase, released a significant amount of the excitotoxic amino acids Asp and Glu from adult rat hippocampal slices incubated in vitro. The concentrations of Asp and Glu in the incubation medium increased by 20 and 30 times respectively when such slices were exposed to simulated ischemic for a 10-min period. However, preparations obtained from 4- to 9-day-old rats did not release Asp or Glu either when exposed to ischemia or after K + depolarization. This release appeared 10–15 days after birth and progressively increased up to 13 months of age. No further increase was observed in 25-month-old animals. The exposure of the slices to a source of oxygen-derived free radicals induced a release of excitotoxic amino acids independently from the age of the rats. The massive excitotoxic amino acid release from adult hippocampal slices and the formation of free radicals induced by ischemic insults has been previously associated with degeneration of hippocampal neurons. The lack of ischemia-induced excitotoxic amino acid release from the newborn hippocampus may help to explain why the newborn hippocampus is more resistant than the adult to hypoxic/ischemic insults.

Brain adenosine and transmitter amino acid release from the ischemic rat cerebral cortex: effects of the adenosine deaminase inhibitor deoxycoformycin.

The effects of a potent adenosine deaminase inhibitor, deoxycoformycin, on purine and amino acid neuro-transmitter release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (20 min) was elicited by four-vessel occlusion. Purine and amino acid releases were compared from control ischemic animals and deoxycoformycin-pretreated ischemic rats. Ischemia enhanced the release of glutamate, aspartate, and gamma-aminobutyric acid into cortical perfusates. The levels of adenosine, inosine, hypoxanthine, and xanthine in the same perfusates were also elevated during and following ischemia. Deoxycoformycin (500 micrograms/kg) enhanced ischemia-evoked release of adenosine, indicating a marked rise in the adenosine content of the interstitial fluid of the cerebral cortex. Inosine, hypoxanthine, and xanthine levels were depressed by deoxycoformycin. Deoxycoformycin pretreatment failed to alter the pattern of amino acid neurotransmitter release from the cerebral cortex in comparison with that observed in control ischemic animals. The failure of deoxycoformycin to attenuate amino acid neurotransmitter release, even though it markedly enhanced adenosine levels in the extracellular space, implies that the amino acid release during ischemia occurs via an adenosine-insensitive mechanism. Inhibition of excitotoxic amino acid release is unlikely to be responsible for the cerebroprotective actions of deoxycoformycin in the ischemic brain.

Effects of thiamine deficiency on brain metabolism: implications for the pathogenesis of the Wernicke-Korsakoff syndrome.

Chronic alcoholism results in thiamine deficiency as a consequence of inadequate dietary intake and of impaired absorption of the vitamin. In addition, there is evidence to suggest that alcohol reduces thiamine phosphorylation to thiamine pyrophosphate (TPP) in brain. TPP is a cofactor for the pyruvate dehydrogenase complex (PDHC), alpha-ketoglutarate dehydrogenase (alpha KGDH) and transketolase (TK), three enzymes involved in cerebral glucose and energy metabolism. Pyrithiamine-induced thiamine deficiency in the rat results in early, selective, reversible reductions of alpha KGDH in brain; PDHC activities are unaffected. Reductions of alpha KGDH are accompanied by decreased aspartate, glutamate and GABA and by concomitantly increased alanine in the brain of thiamine-deficient animals. It is suggested that decreased alpha KGDH, rather than decreased PDHC constitutes 'the biochemical lesion' in thiamine deficiency encephalopathy first enunciated by Peters in the 1930s. If sufficiently prolonged and severe, thiamine deficiency results in brain cell death. Possible mechanisms involved include compromised cerebral energy metabolism and focal accumulation of lactate, both of which could result from decreased activities of alpha KGDH. In addition, it is proposed that brain cell death in severe thiamine deficiency may result from excessive release of excitotoxic amino acids. Comparable mechanisms could be involved in the cell death and in the pathogenesis of the thiamine-unresponsive symptoms of the Wernicke-Korsakoff Syndrome in humans.