Progression in Time of Dentate Gyrus Granule Cell Layer Widening due to Excitotoxicity Occurs along In Vivo LTP Reinstatement and Contextual Fear Memory Recovery.

The dentate gyrus (DG) is the gateway of sensory information arriving from the perforant pathway (PP) to the hippocampus. The adequate integration of incoming information into the DG is paramount in the execution of hippocampal-dependent cognitive functions. An abnormal DG granule cell layer (GCL) widening due to granule cell dispersion has been reported under hyperexcitation conditions in animal models as well as in patients with mesial temporal lobe epilepsy, but also in patients with no apparent relation to epilepsy. Strikingly, it is unclear whether the presence and severity of GCL widening along time affect synaptic processing arising from the PP and alter the performance in hippocampal-mediated behaviors. To evaluate the above, we injected excitotoxic kainic acid (KA) unilaterally into the DG of mice and analyzed the evolution of GCL widening at 10 and 30 days post injection (dpi), while analyzing if KA-induced GCL widening affected in vivo long-term potentiation (LTP) in the PP-DG pathway, as well as the performance in learning and memory through contextual fear conditioning. Our results show that at 10 dpi, when a subtle GCL widening was observed, LTP induction, as well as contextual fear memory, were impaired. However, at 30 dpi when a pronounced increase in GCL widening was found, LTP induction and contextual fear memory were already reestablished. These results highlight the plastic potential of the DG to recover some of its functions despite a major structural alteration such as abnormal GCL widening.

Brainstem activity, apnea, and death during seizures induced by intrahippocampal kainic acid in anaesthetized rats.

OBJECTIVE: To investigate how prolonged seizure activity affects cardiorespiratory function and activity of pre-Bötzinger complex, leading to sudden death. METHODS: Urethane-anesthetized female Long-Evans rats were implanted with nasal thermocouple; venous and arterial cannulae; and electrodes for electrocardiography (ECG) and hippocampal, cortical, and brainstem recording. Kainic acid injection into the ventral hippocampus induced status epilepticus. RESULTS: Seizures caused hypertension, tachycardia, and tachypnea punctuated by recurrent transient apneas. Salivation increased considerably: in 11 of 12 rats, liquid with alkaline pH consistent with saliva was expelled from the mouth. Most transient apneas were obstructive: nasal airflow ceased, while, in 83%, efforts to breathe persisted as continued rhythmic activity of respiratory pre-Bötzinger neurons, inspiratory electromyography (EMG), and excursions of the chest wall and abdomen. Blood pressure oscillated in time with respiratory efforts. This pattern also occurred in a minority of cases (16%) of incomplete apnea, but not in rare cases (1%) of transient central apneas. During transient obstructive apneas, the frequency of all inspiratory efforts decreased abruptly by ~30%, suggesting a resetting of the central respiratory rhythm generator. Twenty-two of thirty-one rats died, due either to obstructive apnea (12) or central apnea following progressive slowing of respiration (10). Most rats dying of central apnea had experienced several transient obstructive apneas. Negative DC field potential shifts of the brainstem followed the final breath, consistent with previous reports on spreading depolarization in mouse models. Timing suggests that the DC shift is a consequence rather than cause of respiratory collapse. Cardiac activity continued for tens of seconds. SIGNIFICANCE: Seizure activity in forebrain induces pronounced autonomic activation and disrupts activity in medullary respiratory centers, resulting in death from either obstructive or central apnea. These results directly inform mechanisms of death in status epilepticus, and indirectly provide clues to mechanisms of sudden unexpected death in epilepsy (SUDEP).

Determination of domoic acid in shellfish extracted by molecularly imprinted polymers.

A selective sample cleanup method using molecularly imprinted polymers was developed for the separation of domoic acid (a shellfish toxin) from shellfish samples. The molecularly imprinted polymers for domoic acid was prepared by emulsion polymerization using 1,3,5-pentanetricarboxylic acid as the template molecule, 4-vinyl pyridine as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, and Span80/Tween-80 (1:1 v/v) as the composite emulsifiers. The molecularly imprinted polymer showed high affinity to domoic acid with a dissociation constant of 13.5 µg/mL and apparent maximum adsorption capacity of 1249 µg/g. They were used as a selective sorbent for the detection of domoic acid from seafood samples coupled with high-performance liquid chromatography. The detection limit of 0.17 µg/g was lower than the maximum level permitted by several authorities. The mean recoveries of domoic acid from clam samples were 93.0-98.7%. It was demonstrated that the proposed method could be applied to the determination of domoic acid from shellfish samples.

Room-temperature fabrication of fluorinated covalent organic polymer @ Attapulgite composite for in-syringe membrane solid-phase extraction and analysis of domoic acid in aquatic products.

A novel fluorinated covalent organic polymer @ attapulgite composite (F-COP@ATP) was prepared at room temperature for in-syringe membrane solid-phase extraction (SM-SPE) of domoic acid (DA) in aquatic products. Natural ore ATP has the advantages of low cost, good mechanical strength and abundant hydroxyl group on its surface, and in-situ modified F-COP layer can provide abundant adsorption sites. F-COP@ATP combining the advantages of F-COP and ATP, becomes an ideal adsorbent for DA extracting. Moreover, a high-throughput sample preparation strategy was carried out by using the F-COP@ATP membrane as syringe filter and assembling syringes with a ten-channel injection pump. In addition, the experimental factors were optimized, such as pH of extract, amount of adsorbent, velocity of extraction and desorption, type and volume of desorption solvent. The DA analytical method was established by SM-SPE-HPLC/tandem mass spectrometry. The method had a wide linear range with low limit of detection (0.344 ng/kg) and low limit of quantification (1.14 ng/kg). F-COP@ATP membrane can be reused more than five times. The method realized the analysis of DA in scallop and razor clam samples, which shows its application prospect in practical analysis. This study provided an efficient, low-energy and mild idea for preparing other reusable natural mineral ATP-based composite materials for separation and enrichment, which reduces the experimental cost and is closer to environmental protection and green chemistry to a certain extent.

Rapid electrochemical detection of domoic acid based on polydopamine/reduced graphene oxide coupled with in-situ imprinted polyacrylamide.

Domoic acid, namely amnesic shellfish toxin, is a highly neurotoxic substance to marine animals and humankind. To reduce the incidence of poisoning accidents, the exploitation of specific and rapid detection method for domoic acid monitoring is highly required. Herein, an electrochemical molecularly imprinted polymer (MIP) sensor based on polydopamine-reduced graphene oxide/polyacrylamide composite (PDA-rGO/PAM) was constructed successfully to detect domoic acid. The domoic acid molecule could be recognized in imprinted cavities of PAM reversibly through hydrogen bonding. PDA-rGO promoted the loading capacity of PAM and improved the charge transfer rate, which amplified the electrical signal response of the MIP sensor. The screen-printed electrode (SPE) modified with PDA-rGO/PAM displayed satisfactory response toward toxin contaminated sample at a linear range from 1 to 600 nM and a low detection limit of 0.31 nM, demonstrating the prospective application of the transducer as a portable sensing platform for the on-site detection of hazardous marine biotoxin. Moreover, benefiting from the superior specificity and stability of MIP, the fabricated sensor could be utilized to detect the domoic acid content in mussel extracts directly without complex pretreatment operation.

Functionalized PEG-PLA nanoparticles for brain targeted delivery of ketoconazole contribute to pregnane X receptor overexpressing in drug-resistant epilepsy.

OBJECTIVE: To develop a functionalized PEG-PLA nanoparticle system containing ketoconazole (KCZ) to overcome the overactivity of pregnane X receptor (PXR) for the treatment of drug-resistant epilepsy (DRE). SIGNIFICANCE: KCZ was developed as a therapy strategy for DRE limited by its lethal hepatotoxicity and minute brain concentration. KCZ-incorporated nanoparticles modified with angiopep-2 (NPs/KCZ) could reduce adverse effects of KCZ and achieve epileptic foci-targeted drug delivery. METHODS: NPs/KCZ was prepared by thin-film hydration method and characterized in vitro and in vivo. The efficacy evaluation of NPs/KCZ was evaluated in a kainic acid (KA)-induced mice model of epilepsy with carbamazepine (CBZ) treatment. RESULTS: The mean particle size and Zeta potential of NPs/KCZ were 17.84 ± 0.33 nm and - 2.28 ± 0.12 mV, respectively. The drug-loading (DL%) of KCZ in nanoparticles was 8.96 ± 0.12 % and the entrapment efficiency (EE%) was 98.56 ± 0.02 %. The critical value of critical micelle concentration was 10-3.3 mg/ml. No obvious cytotoxicity was found in vitro. The behavioral and electrographic seizure activities were obviously attenuated in NPs/KCZ+CBZ group. The CBZ concentration of brain tissues in mice treated with NPs/KCZ+CBZ was significantly increased than those treated with CBZ alone (P = 0.0028). A significantly decreased expression level of PXR and its downstream proteins was observed in NPs/KCZ+CBZ group compared with that in the control and CBZ group (All P < 0.05). CONCLUSION: Our results showed that NPs/KCZ achieved the epileptic foci-targeted delivery of KCZ and ameliorated the efficacy of CBZ on DRE by attenuating the overactivity of PXR.

Molecularly imprinted nanoparticle-based assay (MINA): Potential application for the detection of the neurotoxin domoic acid.

Domoic acid (DA) is a natural amino acid and water-soluble neurotoxic biotoxin primarily produced by the microalgae Pseudo-nitzschia. DA can cause poisoning in humans and a wide variety of marine species. In this work, a molecularly imprinted nanoparticle-based assay (MINA) was developed as an alternative to enzyme-linked immunosorbent assay (ELISA) for selective detection of DA. In contrast with ELISA, MINA uses molecularly imprinted polymer nanoparticles (nanoMIPs) as plastic antibodies due to its higher stability and lower production costs. In this work, dihydrokainic acid (DKA) was used as a dummy template because this molecule is structurally similar to DA but less toxic. The developed MINA had a high linear response for DKA and DA, showing detection limits of 2.12 nmol L-1 and 4.32 nmol L-1, respectively. Additionally, q-RMN studies demonstrated that DKA - nanoMIPs were selective for DKA, since they presented the best association parameters with a high loading load capacity of 175% and an association efficiency of 18%. No cross-reactivity towards 1, 3, 5 - pentanetricarboxylic acid was observed. These results suggest that MINA could be a more robust, more sensitive, and less expensive alternative to ELISA. The assay developed with DKA - nanoMIPs has strong potential for the detection of domoic acid in real samples of red tide.

Study of the neural basis of striatal modulation of the jaw-opening reflex.

Previous experimental data from this laboratory demonstrated the participation of the striatum and dopaminergic pathways in central nociceptive processing. The objective of this study was to examine the possible pathways and neural structures associated with the analgesic action of the striatum. The experiments were carried out in rats anesthetized with urethane. The jaw-opening reflex (JOR) was evoked by electrical stimulation of the tooth pulp of lower incisors and recorded in the anterior belly of the digastric muscles. Intrastriatal microinjection of apomorphine, a nonspecific dopamine agonist, reduced or abolished the JOR amplitude. Electrolytic or kainic acid lesions, unilateral to the apomorphine-injected striatum, of the globus pallidus, substantia nigra pars reticulata, subthalamic nucleus and bilateral lesion the rostroventromedial medulla (RVM), blocked the inhibition of the JOR by striatal stimulation. These findings suggest that the main output nuclei of the striatum and the RVM may be critical elements in the neural pathways mediating the inhibition of the reflex response, evoked in jaw muscles by noxious stimulation of dental pulp.

Organic polymer consumption facilitates domoic acid entry into the marine food web without direct ingestion of Pseudo-nitzschia.

Domoic acid (DA) is a neurotoxin produced by diatoms from the genera Pseudo-nitzschia and Nitzschia. DA is transferred through the food web when consumed by organisms such as copepods (e.g., Acartia tonsa). DA bioaccumulates in higher trophic levels and poses a threat to human health through amnesic shellfish poisoning. Laboratory experiments using a DA reference standard demonstrated that mild turbulence facilitates formation of organic polymer aggregates >0.6 µm in-vivo that can scavenge dissolved DA (dDA). Using A. tonsa, we demonstrate that DA can be assimilated through consumption of these organic polymers which scavenged dDA -a pathway which does not require direct ingestion of the toxin-producer Pseudo-nitzschia. In filtered seawater with spiked DA, copepods accumulated 24.8 ± 4.7 pg DA copepod-1 (2.1 ppm) on average by consuming organic polymers. This was validated in one out of five experiments using ambient DA concentrations. Copepods were suspended in particle-free seawater and accumulated 14.4 ± 3.8 pg DA copepod-1 (1.20 ppm), and in particle-concentrated seawater they accumulated 40.9 ± 3.8 pg DA copepod-1 (3.42 ppm). Data from this experiment suggests that ~34% of the total assimilated DA entered via an organic polymer-bound DA pathway. This experiment had the highest Pseudo-nitzschia spp. abundance (~225,000 cells L - 1) and cellular toxin quota, up to 0.88 pg DA cell-1, relative to the other four ambient DA experiments. These results demonstrate the potential for DA to enter the marine food web through an alternate pathway and may have considerable implications to understanding the flow of DA through marine food webs, and how we monitor DA and its potential vectors into the food web.

Remarkable migration propensity of dental pulp stem cells towards neurodegenerative milieu: An in vitro analysis.

Stem cell therapy provides a ray of hope for treating neurodegenerative diseases (ND). Bone marrow mesenchymal stem cells (BM-MSC) were extensively investigated for their role in neuroregeneration. However, drawbacks like painful bone marrow extraction, less proliferation and poor CNS engraftment following systemic injections of BM-MSC prompt us to search for alternate/appropriate source of MSC for treating ND. In this context, dental pulp stem cells (DPSC) could be an alternative to BM-MSC as it possess both mesenchymal and neural characteristic features due to its origin from ectoderm, ease of isolation, higher proliferation index and better neuroprotection. A study on the migration potential of DPSC compared to BM-MSC in a neurodegenerative condition is warranted. Given the neural crest origin, we hypothesize that DPSC possess better migration towards neurodegenerative milieu as compared to BM-MSC. In this prospect, we investigated the migration potential of DPSC in an in vitro neurodegenerative condition. Towards this, transwell, Matrigel and chorioallantoic membrane (CAM) migration assays were carried-out by seeding hippocampal neurons in the lower chamber and treated with 300 µM kainic acid (KA) for 6 h to induce neurodegeneration. Subsequently, the upper chamber of transwell was loaded with DPSC/BM-MSC and their migration potential was assessed following 24 h of incubation. Our results revealed that the migration potential of DPSC/BM-MSC was comparable in non-degenerative condition. However, following injury the migration potential of DPSC towards the degenerating site was significantly higher as compared to BM-MSC. Furthermore, upon exposure of naïve DPSC/BM-MSCs to culture medium derived from neurodegenerative milieu resulted in significant upregulation of homing factors like SDF-1alpha, CXCR-4, VCAM-1, VLA-4, CD44, MMP-2 suggesting that the superior migration potential of DPSC might be due to prompt expression of homing factors in DPSC compared to BM-MSCs.

Kainate receptors: pharmacology, function and therapeutic potential.

Compared to the other glutamate receptors, progress in the understanding of the functions of kainate receptors (KARs) has lagged behind, due mainly to the relative lack of specific pharmacological tools. Over the last decade subunit selective agonists (e.g. ATPA and 5-iodowillardiine) and orthosteric (e.g. LY382884 and ACET) and allosteric antagonists for KARs that contain GluK1 (GluR5) subunits have been developed. However, no selective ligands for the other KAR subunits have been identified. The use of GluK1 antagonists has enabled several functions of KARs, that contain this subunit, to be identified. Thus, KARs have been shown to regulate excitatory and inhibitory synaptic transmission. In the case of the regulation of L-glutamate release, they can function as facilitatory autoreceptors or inhibitory autoreceptors during repetitive synaptic activation and can respond to ambient levels of L-glutamate to provide a tonic regulation of L-glutamate release. KARs also contribute a component of excitatory synaptic transmission at certain synapses. They can also act as triggers for both long-term potentiation (LTP) and long-term depression (LTD) and rapid alterations in their trafficking can result in altered synaptic transmission during both synaptic plasticity and neuronal development. KARs also contribute to synchronised rhythmic activity in the brain and are involved in forms of learning and memory. With respect to therapeutic indications, antagonists for GluK1 have shown positive activity in animal models of pain, migraine, epilepsy, stroke and anxiety. This potential has now been confirmed in dental pain and migraine in initial studies in man.

Cortical control of appropriate tongue protrusion during licking in cats--increase in regional cerebral blood flow (rCBF) of the contralateral area P and in tongue protrusion after the unilateral area P lesion.

Adequate tongue protrusion may be regulated by cat bilateral area P (the motor cortex for jaw and tongue movements) (Hiraba and Sato, Somatosens Mot Res 2005b;22:183-192). The ICMS (intracortical microstimulation) in the unilateral area P evoked motor effects of tongue protrusion without deviation (Hiraba and Sato, Somatosens Mot Res 2004;23:1-12), and cats with the unilateral lesion of area P showed abnormal tongue protrusion without deviation during licking (Hiraba and Sato, Somatosens Mot Res 2005b;22:183-192). Further, the measurements of the regional cerebral blood flow (rCBF) in the bilateral jaw and tongue motor cortical areas were shown to have the same activity rate during the lateral licking (Hiraba and Sato, Somatosens Mot Res 2005c;22:307-317). We assumed from these results that cortical control for tongue protrusion was executed by networks between the bilateral area P including inhibitory interneurons. We prepared the measurable cats of the rCBF in the contralateral side after the unilateral area P lesion. Changes in the rates of rCBF and tongue protrusion during licking were examined over a long time course of about 1-2 months after the unilateral area P lesion. All cats after the unilateral area P lesion showed increased rate (double or triple in comparison with the normal ones) of rCBF of the contralateral area P in the early (0-20 days) phase. On the other hand, increased rates of tongue protrusion were about 120% in the early phase, and about 180% in the middle (21-35 days) and late (36-last days) phases. The results support the organization of networks between bilateral area P including the inhibitory interneurons.

Studies on mefenamic acid microparticles: formulation, in vitro release, and in situ studies in rats.

In this study, we investigated the in vitro characteristics of mefenamic acid (MA) microparticles as well as their effects on DNA damage. MA-loaded chitosan and alginate beads were prepared by the ionotropic gelation process. Microsponges containing MA and Eudragit RS 100 were prepared by quasi-emulsion solvent diffusion method. The microparticles were characterized in terms of particle size, surface morphology, encapsulation efficiency, and in vitro release profiles. Most of the formulation variables manifested an influence on the physical characteristics of the microparticles at varying degrees. We also studied the effects of MA, MA-loaded microparticles, and three different polymers on rat brain cortex DNA damage. Our results showed that DNA damage was higher in MA-loaded Eudragit microsponges than MA-loaded biodegradable chitosan or alginate microparticles.

Risk assessment of the amnesic shellfish poison, domoic acid, on animals and humans.

Risk assessment of the amnesic shellfish poison, domoic acid, a potent neurotoxin, is evaluated based on its current knowledge and its harmful effects, and is presented under four headings, viz., (1) hazard identification, (2) dose response assessment, (3) exposure assessment and (4) risk characterization. Domoic acid binds the glutamate receptor site of the central nervous system (CNS) of humans and causes depolarization of neurons and an increase in cellularcalcium. In nature, domoic acid is produced by the algae, Pseudonitzschia spp. and they enter into the body of shellfish through their consumption. This toxin is reported to cause gastroenteritis, renal insufficiency confusion and memory loss in humans, since it affects the hippocampus of the brain. In rats, intraperitonial and oral administration of domoic acid result in scratching, tremor and convulsions, and in monkeys, the toxic symptoms like mastication, salivation, projectile vomiting, weakness, teeth grinding and lethargy are apparent. The no-observed-adverse-effect-level (NOAEL) in animals reveals that pure toxin is more effective than those isolated from shellfish. Based on LD50 values, it is found that intraperitonial administration of this toxin in animals is 31 fold more effective than oral administration. Low levels of domoic acid (0.20-0.75 ppm) show no toxic symptoms in non-human primates, but clinical effects are apparent in them and in humans, at a concentration of 1.0 ppm. The tolerable daily intake (TDI) of domoic acid for humans is calculated as 0.075 ppm, whereas for razor clams and crabs, the TDI are 19.4 and 31.5 ppm respectively. The hazard quotient (HQ) is found to be 2. Being an irreversible neurotoxin, domoic acid has severe public health implications. Death occurs in those above 68 years old. In order to ensure adequate protection to public health, the concentration of domoic acid in shellfish and shellfish parts at point of sale shall not exceed the current permissible limit of 20 microg g(-1) tissue. While processing shellfish, it maybe advisable to pay attention to factors such as environmental conditions, inter-organ variability in concentrations of domoic acid and cross contaminations.

Design and development of letrozole nanoemulsion: A comparative evaluation of brain targeted nanoemulsion with free letrozole against status epilepticus and neurodegeneration in mice.

The target of the current study is to formulate letrozole loaded nanoemulsion (LET-NE) for the direct nose to brain delivery to reduce peripheral effects of letrozole (LET). LET-NE is compared against intraperitoneally administered free LET in kainic acid (KA) induced status epilepticus (SE) in mice. LET loaded nanoemulsion (LET-NE) was prepared by aqueous microtitration method using Triacetin, Tween 80 and PEG-400 as the oil phase, surfactant, and co-surfactant. Nanoemulsion was studied for droplet size, polydispersity index (PDI), zeta potential, percentage transmittance, drug content, surface morphology. TEM images of developed formulation demonstrated spherical droplets with a mean diameter of 95.59 ±â€¯2.34 nm, PDI of 0.162 ±â€¯0.012 and zeta potential of -7.12 ±â€¯0.12 mV respectively. In in-vitro and ex-vivo drug release, LET-NE showed prolonged drug release profile as compared to suspension. SE was induced by KA (10 mg/kg, i.p.) in Swiss albino mice. Behavioral seizure monitoring, biochemical estimations, and histopathological examination were performed. The onset time of SE was significantly enhanced and % incidence of SE was reduced by intranasal administration of LET-NE as compared to KA and LET administered intraperitoneally. Biochemical estimations revealed that LET-NE effectively decreased levels of 17-ß estradiol while the levels of 5α-Dihydrotestosterone (5α-DHT) and 3α-androstanediol (3α-Diol) were significantly increased in the hippocampus. In cresyl violet staining LET-NE showed better protection of the hippocampus from neurotoxicity induced by KA as compared to LET. Also, in gamma scintigraphy of mouse brain, intranasal administration of nanoemulsion exhibited the presence of high concentration of LET. The study demonstrates the anticonvulsant and neuroprotective effect of LET-NE probably by inhibition of aromatization of testosterone into 17-ß estradiol, proconvulsant, and diverting the pathway into the synthesis of testosterone metabolites, 3α-Diol with known anticonvulsant and neuroprotective action. Brain targeting of LET-NE showed better anticonvulsant and neuroprotective action than LET.

Mutations to the kainate receptor subunit GluR6 binding pocket that selectively affect domoate binding.

Kainate receptor responses to domoate are characterized by large steady-state currents and slow deactivation kinetics. To improve our understanding of these responses, we mutated residues at the mouth of the agonist binding pocket of GluR6 using whole-cell electrophysiology to characterize the effects of the mutants. We identified two residues where mutations had significant ligand-specific effects. One, Met691, forms a hydrogen bond that seems to facilitate domoate binding by affecting the main-chain conformation. We found that mutation of Met691 to alanine significantly attenuated responses to domoate but had no effect on responses to glutamate, confirming the importance of this main-chain interaction in GluR6. The second residue, Val685, is located at the mouth of the binding pocket, adjacent to the domoate side-arm. Mutation of Val685 to glutamine increased the rate of decay from steady-state responses to domoate by more than 50-fold but had no effect on the rate or extent of desensitization or on the kinetics of responses to either glutamate or kainate. The V685Q mutant also significantly reduced the potencies of both glutamate (peak) and domoate (peak and steady-state). Empirical analysis using a basic kinetic model indicated that the V685Q phenotype could be fully explained by faster ligand dissociation. The V685Q mutant accelerated receptor deactivation without affecting either desensitization or gating, making it a potentially useful tool for further dissection of ligand binding and gating in kainate receptors.

A highly sulfated chondroitin sulfate preparation, CS-E, prevents excitatory amino acid-induced neuronal cell death.

Chondroitin sulfate (CS) is a major microenvironmental molecule in the CNS, and there have been few reports about its neuroprotective activity. As neuronal cell death by excitotoxicity is a crucial phase in many neuronal diseases, we examined the effect of various CS preparations on neuronal cell death induced by the excitotoxicity of glutamate analogs. CS preparations were added to cultured neurons before and after the administration of glutamate analogs. Then, the extents of both neuronal cell death and survival were estimated. Pre-administration of a highly sulfated CS preparation, CS-E, significantly reduced neuronal cell death induced by not only NMDA but also (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate. Neither CS preparations other than CS-E nor other highly sulfated polysaccharides such as heparin and dextran sulfate exerted any neuroprotective effects. NMDA-induced current in neurons was not changed by pre-administration of CS-E, but the pattern of protein-tyrosine phosphorylation was changed. In addition, the elevation of caspase 3 activity was significantly suppressed in CS-E-treated neurons. These results indicate that CS-E prevents neuronal cell death mediated by various glutamate receptors, and suggest that phosphorylation-related intracellular signals and the suppression of caspase 3 activation are implicated in neuroprotection by CS-E.

Applying molecular modelling and experimental studies to develop molecularly imprinted polymer for domoic acid enrichment from both seawater and shellfish.

A highly selective sample cleanup method using molecularly imprinted polymers (MIP) was developed for the enrichment of domoic acid (DA, an amnesic shellfish toxin) from both seawater and shellfish samples. Molecular modelling was firstly applied to screening a suitable functional monomer and optimize the polymer preparation. Theoretical results were in a good agreement with those of the experimental studies. MIP was prepared by precipitation polymerization using 1, 3, 5-pentanetricarboxylic acid and 2-(Trifluoromethyl)acrylic acid as the template molecule and functional monomer, respectively. The morphology and molecular structure of MIP were revealed by scanning electron microscope (SEM) and fourier transform infrared spectroscopy (FTIR), respectively. The obtained MIP showed high affinity and selectivity for DA with binding site numbers of 0.875 mg g-1 and an average association constant of 0.219 L mg-1 evaluated by adsorption experiments. The developed molecularly imprinted solid-phase extraction (MISPE) column achieved satisfied adsorption rate (99.2%) and recovery (71.2%) with relative standard deviation (RSD) less than 1.0%, which is more stable and precise than the C18, SAX, and HLB columns. Finally, the determination method for DA in both seawater and shellfish samples was then successfully established and validated using MISPE coupled with high-performance liquid chromatography-ultraviolet detection (HPLC-UV). The method limit of detection was 20 µg L-1 and 50 µg kg-1 for seawater and shellfish, respectively. This study demonstrates that molecular modelling is a useful tool to screening functional monomer and optimize polymer preparation. It provides an innovative polymer for trace DA monitoring in both seawater and shellfish.

Acid reflux induced laryngospasm as a potential mechanism of sudden death in epilepsy.

OBJECTIVE: Recent research suggests that obstructive laryngospasm and consequent respiratory arrest may be a mechanism in sudden unexpected death in epilepsy. We sought to test a new hypothesis that this laryngospasm is caused by seizures driving reflux of stomach acid into the larynx, rather than spontaneous pathological activity in the recurrent laryngeal nerve. APPROACH: We used an acute kainic acid model under urethane anesthesia to observe seizure activity in Long-Evans rats. We measured the pH in the esophagus and respiratory activity. In a subset of experiments, we blocked acid movement up the esophagus with a balloon catheter. MAIN RESULTS: In all cases of sudden death, terminal apnea was preceded by a large pH drop from 7 to 2 in the esophagus. In several animals we observed acidic fluid exiting the mouth, sometimes in large quantities. In animals where acid movement was blocked, sudden deaths did not occur. No acid was detected in controls. SIGNIFICANCE: The results suggest that acid movement up the esophagus is a trigger for sudden death in KA induced seizures. The fact that blocking acid also eliminates sudden death implies causation. These results may provide insight to the mechanism of SUDEP in humans.

Multipoint recognition of domoic acid from seawater by dummy template molecularly imprinted solid-phase extraction coupled with high-performance liquid chromatography.

Due to the high cost of domoic acid (DA), different carboxylic acid compounds including indole-3-acetic acid (IAA), pyrrole-2-formic acid (PFA), pyridine-2,3-dicarboxylic anhydride (PDA), trimesitinic acid (TA) and citric acid (CA) were investigated as dummy templates for the molecularly imprinted solid-phase extraction (MISPE) for selective isolation and pre-concentration of an amnesic shellfish poison (ASP), DA. The highest binding amount of the polymers towards DA was obtained when CA was used as dummy template owing to its high hydrophilicity. In addition, the "four-point" recognition site constructed by three COOH groups and a OH group in CA was also speculated to be the reason for the high binding amount of CA-MIPs and the rebinding of DA can be depend on the three COOH groups and a NH group with conformational change in the recognition process. Finally, the CA-MISPE column was chosen for DA isolation and pre-concentration and effective result was obtained with recoveries higher than 90% and relative standard deviation (RSD) less than 5% (n=3). This new polymer can be effectively applied to the monitoring and predicting the existence of trace DA.