Capsaicin protects cortical neurons against ischemia/reperfusion injury via down-regulating NMDA receptors.

Capsaicin, the ingredient responsible for the pungent taste of hot chili peppers, is widely used in the study and management of pain. Recently, its neuroprotective effect has been described in multiple studies. Herein, we investigated the underlying mechanisms for the neuroprotective effect of capsaicin. Direct injection of capsaicin (1 or 3nmol) into the peri-infarct area reduced the infarct volume and improved neurological behavioral scoring and motor coordination function in the middle cerebral artery occlusion (MCAO)/reperfusion model in rats. The time window of the protective effect of capsaicin was within 1h after reperfusion, when excitotoxicity is the main reason of cell death. In cultured cortical neurons, administration of capsaicin attenuated glutamate-induced excitotoxic injury. With respect to the mechanisms of the neuroprotective effect of capsaicin, reduced calcium influx after glutamate stimulation was observed following capsaicin pretreatment in cortical neurons. Trpv1 knock-out abolished the inhibitory effect of capsaicin on glutamate-induced calcium influx and subsequent neuronal death. Reduced expression of GluN1 and GluN2B, subunits of NMDA receptor, was examined after capsaicin treatment in cortical neurons. In summary, our studies reveal that the neuroprotective effect of capsaicin in cortical neurons is TRPV1-dependent and down-regulation of the expression and function of NMDA receptors contributes to the protection afforded by capsaicin.

Effects of Propofol on Excitatory and Inhibitory Amino Acid Neurotransmitter Balance in Rats with Neurogenic Pulmonary Edema Induced by Subarachnoid Hemorrhage.

INTRODUCTION: Propofol exhibits neuroprotective effects mediated by the inhibition of excitatory amino acid (EAA) neurotransmitter release and potentiation of inhibitory amino acid (IAA) neurotransmitters. To our knowledge, this is the first study to investigate the effects of propofol on the EAA and IAA balance in neurogenic pulmonary edema (NPE). METHODS: Sixty male Wistar rats were randomized to Sham, NPE, Low-dose propofol, and High-dose propofol groups. NPE was induced via rapid injection of autologous blood (0.5 ml) into the cisterna magna. The Low- and High-dose propofol groups were pretreated with boluses of 2 and 5 mg kg(-1), respectively, prior to blood injection, followed by continuous propofol infusion at 6 and 15 mg kg(-1) h(-1), respectively. The mean arterial pressure (MAP), heart rate, intracranial pressure (ICP), peak inspiratory pressure (PIP), and arterial blood gases were continuously recorded. After 2 h, the lung wet-to-dry weight ratio, total protein concentration in the bronchoalveolar lavage fluid (BALF), brain water content, cortical EAA and IAA levels, chest X-ray, and histological staining of lung sections were evaluated. RESULTS: Blood injections into the cisterna magna induced NPE and hemodynamic changes. Propofol alleviated the increases in the MAP, ICP, and PIP, improved oxygenation and histopathological changes, ameliorated pulmonary and cerebral edema, increased the IAA brain levels, and decreased the ratio of Glu to γ-aminobutyric acid. CONCLUSIONS: The current findings suggest that propofol improves NPE likely via IAA accumulation and the regulation of EAA and IAA balance, which may represent an effective treatment for NPE.

A review of experimental research on herbal compounds in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease worldwide, leading to progressive muscle atrophy and paralysis. The limited success of conventional treatment for ALS has prompted investigations into complementary and alternative therapies. Herbal remedies provide good prospects of ALS prevention and treatment, with advantages such as multiple targets, multiple links, and few side effects. Studies in vitro and in vivo have shown that herbs have a great potential for treatment of ALS, with therapeutic effects against oxidative stress, excitatory amino acid toxicity, neuroinflammation, and calcium cytotoxicity. Active monomers or ingredients extracted from herbs are considered promising candidates for ALS. Therefore, we review recent experimental research on monomers and compounds isolated from herbal remedies for preventing and treating ALS.

TGF-ß1 attenuates spinal neuroinflammation and the excitatory amino acid system in rats with neuropathic pain.

UNLABELLED: Previous studies have reported that the intrathecal (i.t.) administration of transforming growth factor ß1 (TGF-ß1) prevents and reverses neuropathic pain. However, only limited information is available regarding the possible role and effects of spinal TGF-ß1 in neuropathic pain. We aimed to investigate the antinociceptive effects of exogenous TGF-ß1 on chronic constriction injury (CCI)-induced neuropathic pain in rats. We demonstrated that sciatic nerve injury caused a downregulation of endogenous TGF-ß1 levels on the ipsilateral side of the lumbar spinal dorsal gray matter, and that the i.t. administration of TGF-ß1 (.01-10 ng) significantly attenuated CCI-induced thermal hyperalgesia in neuropathic rats. TGF-ß1 significantly inhibited CCI-induced spinal neuroinflammation, microglial and astrocytic activation, and upregulation of tumor necrosis factor-α. Moreover, i.t. TGF-ß1 significantly attenuated the CCI-induced downregulation of glutamate transporter 1, the glutamate aspartate transporter, and the excitatory amino acid carrier 1 on the ipsilateral side. Furthermore, i.t. TGF-ß1 significantly decreased the concentrations of 2 excitatory amino acids, aspartate and glutamate, in the spinal dialysates in CCI rats. In summary, we conclude that the mechanisms of the antinociceptive effects of i.t. TGF-ß1 in neuropathy may include attenuation of spinal neuroinflammation, attenuation, or upregulation of glutamate transporter downregulation, and a decrease of spinal extracellular excitatory amino acids. PERSPECTIVE: Clinically, medical treatment is usually initiated after the onset of intractable pain. Therefore, in the present study, i.t. TGF-ß1 was designed to be administered 2 weeks after the establishment of CCI pain. Compared to the continuous TGF-ß1 infusion mode, single-dose administration seems more convenient and practical to use.

Purinergic P2X receptor regulates N-methyl-D-aspartate receptor expression and synaptic excitatory amino acid concentration in morphine-tolerant rats.

BACKGROUND: The present study examined the effect of P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) on morphine tolerance in rats. METHODS: Male Wistar rats were implanted with two intrathecal catheters with or without a microdialysis probe, then received a continuous intrathecal infusion of saline (control) or morphine (tolerance induction) for 5 days. RESULTS: Long-term morphine infusion induced antinociceptive tolerance and up-regulated N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in both total lysate and synaptosome fraction of the spinal cord dorsal horn. TNP-ATP (50 µg) treatment potentiated the antinociceptive effect of morphine, with a 5.5-fold leftward shift of the morphine dose-response curve in morphine-tolerant rats, and this was associated with reversal of the up-regulated NR1 and NR2B subunits in the synaptosome fraction. NR1/NR2B-specific antagonist ifenprodil treatment produced a similar effect as TNP-ATP; it also potentiated the antinociceptive effect of morphine. On day 5, morphine challenge resulted in a significant increase in aspartate and glutamate concentration in the cerebrospinal fluid dialysates of morphine-tolerant rats, and this effect was reversed by TNP-ATP treatment. Moreover, the amount of immunoprecipitated postsynaptic density-95/NR1/NR2B complex was increased in morphine-tolerant rats, and this was prevented by the TNP-ATP treatment. CONCLUSIONS: The findings suggest that attenuation of morphine tolerance by TNP-ATP is attributed to down-regulation of N-methyl-d-aspartate receptor subunits NR1 and NR2B expression in the synaptosomal membrane and inhibition of excitatory amino acids release in morphine-tolerant rats. The TNP-ATP regulation on the N-methyl-d-aspartate receptor expression may be involved in a loss of scaffolding proteins postsynaptic density-95.

Bromocriptine, an ergot alkaloid, inhibits excitatory amino acid release mediated by glutamate transporter reversal.

Bromocriptine, a dopamine D(2) receptor agonist, has widely been used for patients with Parkinson's disease. The aim of the present study was to investigate the effect of bromocriptine on glutamate transporter. Since the astroglial glutamate transporter GLT-1 (EAAT2) is the predominant isoform in the forebrain, we generated EAAT2-expressing human embryonic kidney cells and immortalized mouse astrocytes. In the present studies, we observed a GLT-1-immunoreactive band and significant Na(+)-dependent d-[(3)H] aspartate uptake. Furthermore, the glutamate transporter inhibitors, dl-threo-beta-benzyloxyaspartic acid (TBOA) and dihydrokainate (DHK), displayed a dose-dependent reduction of d-[(3)H] aspartate uptake in both types of cells. In contrast, cells exposed to either chemical anoxia or high KCl elicited a marked release of d-[(3)H] aspartate, and the release was inhibited by TBOA and DHK, implying the contribution of glutamate transporter reversal. Interestingly, we found that bromocriptine dose-dependently inhibits d-[(3)H] aspartate release elicited by chemical anoxia or high KCl, while no changes occurred in the uptake. The inhibitory action of bromocriptine was not affected by sulpiride, a dopamine D(2) receptor antagonist. On the other hand, bromocriptine had no effect on swelling-induced d-[(3)H] aspartate release, which is mediated by volume-regulated anion channels. In vivo studies revealed that bromocriptine suppresses the excessive elevation of glutamate levels in gerbils subjected to transient forebrain ischemia in a manner similar to DHK. Taken together, these results provide evidence that bromocriptine inhibits excitatory amino acid release via reversed operation of GLT-1 without altering forward transport.

Anti-apoptotic effect of esculin on dopamine-induced cytotoxicity in the human neuroblastoma SH-SY5Y cell line.

Dopamine (DA), as a neurotoxin, can elicit severe Parkinson's disease-like syndrome by elevating intracellular reactive oxygen species (ROS) levels and apoptotic activity. In this study, we examined the effect of esculin, which was extracted from Fraxinus sielboldiana blume, on DA-induced cytotoxicity and the underlying mechanism in human neuroblastoma SH-SY5Y cells. Our results suggest that the protective effects of esculin (10(-7), 10(-6) and 10(-5) M) on DA-induced cytotoxicity may be ascribed to its anti-oxidative properties by reducing ROS level, and its anti-apoptotic effect via protecting mitochondrion membrane potential (DeltaPsim), enhancing superoxide dismutaese (SOD) activity and reduced glutathione (GSH) levels, and regulating P53, Bax and Bcl-2 expression. In addition, esculin inhibited the release of cytochrome c and apoptosis-inducing factor (AIF), and the protein expression of activated caspase 3. These data indicate that esculin may provide a useful therapeutic strategy for the treatment of progressive neurodegenerative diseases such as Parkinson's disease (PD).

Cannabinoids inhibit sodium-dependent, high-affinity excitatory amino acid transport in cultured rat cortical astrocytes.

Cannabinoids have been shown to increase the extracellular levels of glutamate in vivo and in vitro, but no studies have evaluated the possible involvement of glial glutamate reuptake system. The present study investigates whether cannabinoids and endocannabinoid, anandamide have an effect on astroglial excitatory amino acid (EAA) transport. The kinetics of glutamate transport was studied in rat cortical astrocytes, using the radiolabeled, non-metabolized amino acid, D-[3H] aspartate in the absence or presence of cannabinoid receptor agonists. The results show that in vehicle controls the uptake of d-aspartate was rapid, sodium-dependent and saturated within the first 5 min, resulting in a K(m) 7.365+/-1.16 micromol/L (n=5) and the maximum velocity (V(max)) 1207+/-51 nmol/mg protein/min. Addition of the synthetic cannabinoid analog R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol][1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2; 3 micromol/L) increased the K(m) (26.25+/-4.84 micromol/L) without affecting the V(max) (1122+/-77 nmol/mg protein/min), suggesting the inhibition was competitive and reversible. Various other cannabinoid agonists also inhibited D-aspartate uptake in a dose-dependent and stereospecific manner. The cannabinoid inhibition of EAA transport was partially blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 100 nmol/L). The inhibitory effects of WIN 55,212-2, or its endogenous counterpart anandamide were reversed by 98,059, an inhibitor of mitogen-activated kinase (MAPK) kinase (MEK). These results suggest that cannabinoids and endocannabinoids may constitute a novel class of inhibitors of astroglial glutamate transport system.

[Neuroprotection in brain ischemia--doubts and hopes]. / Neuroprotekció agyi ischaemiában--kételyek és remények.

In ischaemic stroke the two major potential therapeutic strategies are aimed at either improving cerebral blood flow or directly interacting with the cytotoxic cascade--a large body of evidence gained from animal studies is in support of them. In clinical trials direct neuroprotection by blocking the neurotoxic cascade remained ineffective, although there are several clinical trials still in progress. We summarize the experimental data and present the results of clinical trials and also discuss why so many drugs, which were effective in animal studies, failed in human trials. It is emphasized, that 1. in most animal studies the reduction of infarct size, i.e. the amount of saved penumbral tissue, was the outcome measure, whereas neurological function remained unassessed; 2. the recovery of intellectual performance and higher cortical functions are of major importance in the future quality of life in stroke victims; however, it is impossible to examine these parameters appropriately in animal studies; 3. in many clinical trials the patient population was rather heterogenous and low in number, the study protocol was not optimal and the critical analysis of the subacute and chronic phase was lacking or insufficient. We present the major experimental stroke models, discuss their similarities, differences and limitations as compared to the human pathophysiological processes. The pitfalls of extrapolating data from animal studies to clinical practice are also summarized. The complex network of functional and morphological intercellular connections, the long timescale of neurotoxic and reparative events and the lessons learned from clinical trials suggest, that the use of drug combinations (therapeutic cocktails) targeting multiple steps of the neurotoxic cascade would hopefully result in more effective treatment of ischaemic stroke. Strategies to facilitate brain plasticity and regeneration is an additional promising tool to enhance recovery in brain ischaemia.

Inhibition of release of taurine and excitatory amino acids in ischemia and neuroprotection.

Volume regulated anion channels (VRAC) have been extensively studied in purified single cell systems like cell cultures where they can be activated by cell swelling. This provides a convenient way of analyzing mechanisms and will likely lead to the holy grails of the field, namely the nature or natures of the volume sensor and the nature or natures of VRACs. Important reasons for such an understanding are that these channels are ubiquitous and have important physiological functions which under pathological conditions convert to deleterious effects. Here we summarize data showing the involvement of VRACs in ischemia-induced release of excitatory amino acids (EAAs) in a rat model of global ischemia. Using microdialysis studies we found that reversal of the astrocytic glutamate transporter and VRACs contribute about equally to the large initial release of EAAs and together account for around 80% of the total release. We used the very potent VRAC blocker, tamoxifen, to see if such inhibition of EAA release via VRACs led to significant neuroprotection. Treatment in the focal rat MCA occlusion model led to around 80% reduction in infarct size with an effective post initiation of ischemia therapeutic window of three hours. However, the common problem of other effects for even the most potent inhibitors pertains here, as tamoxifen has other, potentially neuroprotective, effects. Thus it inhibits nitrotyrosine formation, likely due to its inhibition of nNOS and reduction of peroxynitrite formation. Although tamoxifen cannot therefore be used as a test of the "VRAC-excitotxicity" hypothesis it may prove successful for translation of basic stroke research to the clinic because of its multiple targets.

[Research progress of neuroprotective mechanisms of Gastrodia elata and its preparation].

The effects of Gastrodia elata on preventing decrepitude and advancing memory are closely associated with its neuroprotective activity. Previous researches proved that G. elata, its active components and preparations played a neuroprotective role by affecting the excitotoxicity, nitric monoxide (NO) system, neuroglia, biomembrane, oxidative neurotoxicity, apoptosis et al. Recent researches also suggest that reducing energy metabolism impairment, anti-inflammatory and immune modulating function may be new research targets of neuroprotective mechanism of G. elata.

"In vivo" monitoring of neuronal network activity in zebrafish by two-photon Ca(2+) imaging.

The zebrafish larva is a powerful model for the analysis of behaviour and the underlying neuronal network activity during early stages of development. Here we employ a new approach of "in vivo" Ca(2+) imaging in this preparation. We demonstrate that bolus injection of membrane-permeable Ca(2+) indicator dyes into the spinal cord of zebrafish larvae results in rapid staining of essentially the entire spinal cord. Using two-photon imaging, we could monitor Ca(2+) signals simultaneously from a large population of spinal neurons with single-cell resolution. To test the method, Ca(2+) transients were produced by iontophoretic application of glutamate and, as observed for the first time in a living preparation, of GABA or glycine. Glycine-evoked Ca(2+) transients were blocked by the application of strychnine. Sensory stimuli that trigger escape reflexes in mobile zebrafish evoked Ca(2+) transients in distinct neurons of the spinal network. Moreover, long-term recordings revealed spontaneous Ca(2+) transients in individual spinal neurons. Frequently, this activity occurred synchronously among many neurons in the network. In conclusion, the new approach permits a reliable analysis with single-cell resolution of the functional organisation of developing neuronal networks.

Emerging molecular targets in the treatment of bacterial meningitis.

The mortality and morbidity associated with bacterial meningitis have remained significant despite advances in antimicrobial chemotherapy and supportive care. A major contributing factor to this high mortality and morbidity is our incomplete understanding of the pathogenesis of this disease and its associated neurological sequelae. Most cases of bacterial meningitis develop as a result of haematogenous spread, but it is unclear how circulating bacteria cross the blood-brain barrier. Experimental animal studies indicate that two forms of neuronal injury, such as necrotic cortical injury and apoptotic hippocampal injury, are predominant in bacterial meningitis, but the mechanisms by which these two forms of injury occur are unclear. Recent studies have identified several bacteria-host determinants for bacterial translocation of the blood-brain barrier, and several host inflammatory markers that are associated with neuronal injury in animal models of experimental bacterial meningitis. These determinants/markers may provide important targets for the prevention and treatment of bacterial meningitis. This review focuses on representative steps in the pathogenesis of bacterial meningitis that are likely to be key targets in coming years, and summarises the status of current knowledge for each target.

Effects of acamprosate on excitatory amino acids during multiple ethanol withdrawal periods.

BACKGROUND: Our previous studies on the effects of acamprosate on enhanced locomotion during repeated withdrawals are now extended to the effects of acamprosate on excitatory amino acids in the hippocampus during repeated ethanol withdrawals. METHODS: In this study, Wistar rats were made ethanol dependent by 4 weeks of vapor inhalation. After this first cycle of chronic ethanol treatment, rats underwent repeated and alternate cycles of 24 hr withdrawals and 1 week of chronic ethanol treatment. The microdialysis technique was used together with high-performance liquid chromatography and electrochemical detection to quantify different amino acids such as aspartate and glutamate. RESULTS: An intraperitoneal administration of acamprosate (400 mg/kg) to naïve rats did not alter aspartate or glutamate levels compared with the saline groups. During the first cycle of ethanol withdrawal, the administration of acamprosate (400 mg/kg, intraperitoneally) 2 hr after the commencement of ethanol withdrawal decreased both aspartate and glutamate microdialysate levels when compared with their respective saline group. Acamprosate administration also significantly decreased glutamate levels during the third withdrawal compared with the saline group, whereas no changes were seen in aspartate levels. CONCLUSION: The results of this work demonstrate that acamprosate reduced the excitatory amino acid glutamate increase observed during repeated ethanol withdrawal. These effects of acamprosate may provide a protective mechanism against neurotoxicity by reducing excitatory amino acids, particularly glutamate.

Neuroprotective agents in acute ischemic stroke.

The concept of neuroprotection relies on the principle that delayed neuronal injury occurs after ischemia. The phenomenon of the "ischemic cascade" has been described, and each step along this cascade provides a target for therapeutic intervention. A wide variety of drugs have been studied in humans. Ten classes of neuroprotective agents have reached phase III efficacy trials but have shown mixed results. They included calcium channel antagonists, NMDA receptor antagonists, lubeluzole, CDP-choline, the free radical scavenger tirilazad and ebselen, enlimomab, GABA agonist clomethiazole, the sodium channel antagonist fosphenytoin, magnesium, glycine site antagonist GV150526 and piracetam. Furthermore, the mechanisms that underlie the development of focal ischemic injury continue to be discovered, opening new therapeutic perspective for neuroprotection that might clinically be applicable in the future.

Neuronal injury in bacterial meningitis: mechanisms and implications for therapy.

In bacterial meningitis, long-term neurological sequelae and death are caused jointly by several factors: (1) the systemic inflammatory response of the host, leading to leukocyte extravasation into the subarachnoid space, vasculitis, brain edema and secondary ischemia; (2) stimulation of resident microglia within the CNS by bacterial compounds; and (3) possible direct toxicity of bacterial compounds on neurons. Neuronal injury is mediated by the release of reactive oxygen intermediates, proteases, cytokines and excitatory amino acids, and is executed by the activation of transcription factors, caspases and other proteases. In experimental meningitis, dexamethasone as an adjunct to antibiotic treatment leads to an aggravation of neuronal damage in the hippocampal formation, suggesting that corticosteroids might not be the ideal adjunctive therapy. Several approaches that interfere selectively with the mechanisms of neuronal injury are effective in animal models, including the use of nonbacteriolytic protein synthesis-inhibiting antibiotics, antioxidants and inhibitors of transcription factors, matrix metalloproteinases, and caspases.

[Medicinal and chemical approaches to focused search of agents for treatment and therapy of Alzheimer disease]. / Mediko-khimicheskie podkhody k napravlennomu poisku preparatov dlia lecheniia i preduprezhdeniia bolezni Altsgeimera.

The analysis and justification of medicinal chemistry approaches for focused search of novel agents for Alzheimer's disease (AD) and related disorders treatment and prevention have been reviewed. The systematization of modern biochemical and structural date related to the action of physiologically active compounds on the nervous system apparatus engaged in the AD-like disorders pathogenesis was performed. The major attention was paid to the cholinomimetic, anti-amyloid and antimetabolic approaches, basing on the results published in scientific literature in 3-4 last years and results of preclinical and clinical trials, presented in the internet database in the fall of 2000.

Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells.

Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases. The present study was performed to evaluate the inhibitory effect of fangchinoline, a bis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline (1 and 5 microM) inhibited glutamate (1 mM), N-methyl-D-aspartate (NMDA; 1 mM) and kainate (100 microM)-induced neuronal cell death which was measured by trypan blue exclusion test. Fangchinoline (1 and 5 microM) inhibited glutamate release into medium induced by NMDA (1 mM) and kainate (100 microM), which was measured by HPLC. And fangchinoline (5 microM) inhibited glutamate (1 mM)-induced elevation of intracellular calcium concentration. These results suggest that inhibition of Ca2+ influx by fangchinoline may contribute to the beneficial effects on neurodegenerative effect of glutamate in pathophysiological conditions.