Gene therapy for epilepsy targeting neuropeptide Y and its Y2 receptor to dentate gyrus granule cells.

Gene therapy is emerging as an alternative option for individuals with drug-resistant focal epilepsy. Here, we explore the potential of a novel gene therapy based on Neuropeptide Y (NPY), a well-known endogenous anticonvulsant. We develop a lentiviral vector co-expressing NPY with its inhibitory receptor Y2 in which, for the first time, both transgenes are placed under the control of the minimal CamKIIa(0.4) promoter, biasing expression toward excitatory neurons and allowing autoregulation of neuronal excitability by Y2 receptor-mediated inhibition. Vector-induced NPY and Y2 expression and safety are first assessed in cultures of hippocampal neurons. In vivo experiments demonstrate efficient and nearly selective overexpression of both genes in granule cell mossy fiber terminals following vector administration in the dentate gyrus. Telemetry video-EEG monitoring reveals a reduction in the frequency and duration of seizures in the synapsin triple KO model. This study shows that targeting a small subset of neurons (hippocampal granule cells) with a combined overexpression of NPY and Y2 receptor is sufficient to reduce the occurrence of spontaneous seizures.

Circulating microRNAs and isomiRs as biomarkers for the initial insult and epileptogenesis in four experimental epilepsy models: The EPITARGET study.

OBJECTIVE: Structural epilepsies can manifest months or years after the occurrence of an initial epileptogenic insult, making them amenable for secondary prevention. However, development of preventive treatments has been challenged by a lack of biomarkers for identifying the subset of individuals with the highest risk of epilepsy after the epileptogenic insult. METHODS: Four different rat models of epileptogenesis were investigated to identify differentially expressed circulating microRNA (miRNA) and isomiR profiles as biomarkers for epileptogenesis. Plasma samples were collected on day 2 and day 9 during the latency period from animals that did or did not develop epilepsy during long-term video-electroencephalographic monitoring. miRNAs and isomiRs were identified and measured in an unsupervised manner, using a genome-wide small RNA sequencing platform. Receiver operating characteristic analysis was performed to determine the performance of putative biomarkers. RESULTS: Two days after an epileptogenic insult, alterations in the levels of several plasma miRNAs and isomiRs predicted epileptogenesis in a model-specific manner. One miRNA, miR-3085, showed good sensitivity (but low specificity) as a prognostic biomarker for epileptogenesis in all four models (area under the curve = .729, sensitivity = 83%, specificity = 64%, p < .05). SIGNIFICANCE: Identified plasma miRNAs and isomiRs are mostly etiology-specific rather than common prognostic biomarkers of epileptogenesis. These data imply that in preclinical and clinical studies, it may be necessary to identify specific biomarkers for different epilepsy etiologies. Importantly, circulating miRNAs like miR-3085 with high negative predictive value for epileptogenesis in different etiologies could be useful candidates for initial screening purposes of epileptogenesis risk.

Lack of Direct Effects of Neurotrophic Factors in an In Vitro Model of Neuroinflammation.

Neuroinflammation is associated with several neurological disorders including temporal lobe epilepsy. Seizures themselves can induce neuroinflammation. In an in vivo model of epilepsy, the supplementation of brain-derived neurotropic factor (BDNF) and fibroblast growth factor-2 (FGF-2) using a Herpes-based vector reduced epileptogenesis-associated neuroinflammation. The aim of this study was to test whether the attenuation of the neuroinflammation obtained in vivo with BDNF and FGF-2 was direct or secondary to other effects, for example, the reduction in the severity and frequency of spontaneous recurrent seizures. An in vitro model of neuroinflammation induced by lipopolysaccharide (LPS, 100 ng/mL) in a mouse primary mixed glial culture was used. The releases of cytokines and NO were analyzed via ELISA and Griess assay, respectively. The effects of LPS and neurotrophic factors on cell viability were determined by performing an MTT assay. BDNF and FGF-2 were tested alone and co-administered. LPS induced a significant increase in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and NO. BDNF, FGF-2, and their co-administration did not counteract these LPS effects. Our study suggests that the anti-inflammatory effect of BDNF and FGF-2 in vivo in the epilepsy model was indirect and likely due to a reduction in seizure frequency and severity.

Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression.

Inactivation of all herpes simplex virus (HSV) immediate early (IE) genes to eliminate vector cytotoxicity results in rapid silencing of the viral genome, similar to the establishment of HSV latency. We recently reported that silencing of a nonviral reporter cassette could be overcome in nonneuronal cells by positioning the cassette in the viral latency (LAT) locus between resident chromatin boundary elements. Here, we tested the abilities of the chicken hypersensitive site 4 insulator and the human ubiquitous chromatin opening element A2UCOE to promote transgene expression from an IE-gene-inactivated HSV vector. We found that A2UCOE was particularly active in nonneuronal cells and reduced reporter promoter occupancy by a repressive histone mark. We determined whether multiple transgenes could be expressed under the control of different promoters from different loci of the same virus. The results showed abundant coexpression of LAT-embedded and A2UCOE-flanked genes in nonneuronal cells. In addition, a third reporter gene without known protective elements was active in cultured rat sensory neurons. These findings indicate that cellular antisilencing sequences can contribute to the expression of multiple genes from separate promoters in fully IE gene-disabled HSV vectors, providing an opportunity for therapeutic applications requiring mutually independent expression of different gene products from a single vector.IMPORTANCE Gene therapy has now entered a phase of development in which a growing number of recessive single gene defects can be successfully treated by vector-mediated introduction of a wild-type copy of the gene into the appropriate tissue. However, many disease conditions, such as neurodegeneration, cancer, and inflammatory processes, are more complex, requiring either multiple gene corrections or provision of coordinated gene activities to achieve a therapeutic outcome. Although herpes simplex virus (HSV) vectors have the capacity to meet this need, the challenge has been to genetically engineer the HSV genome in a manner to prevent expression of any viral genes while retaining the ability to express multiple therapeutic transgenes under independent transcriptional control. Here, we show that non-HSV insulator elements can be applied to retain at least transient transgene activity from multiple viral loci, thereby opening the door for more complex gene therapy applications in the future.

Cognitive comorbidities in the rat pilocarpine model of epilepsy.

Patients with epilepsy are prone to cognitive decline, depression, anxiety and other behavioral disorders. Cognitive comorbidities are particularly common and well-characterized in people with temporal lobe epilepsy, while inconsistently addressed in epileptic animals. Therefore, the aim of this study was to ascertain whether there is good evidence of cognitive comorbidities in animal models of epilepsy, in particular in the rat pilocarpine model of temporal lobe epilepsy. We searched the literature published between 1990 and 2023. The association of spontaneous recurrent seizures induced by pilocarpine with cognitive alterations has been evaluated by using various tests: contextual fear conditioning (CFC), novel object recognition (NOR), radial and T-maze, Morris water maze (MWM) and their variants. Combination of results was difficult because of differences in methodological standards, in number of animals employed, and in outcome measures. Taken together, however, the analysis confirmed that pilocarpine-induced epilepsy has an effect on cognition in rats, and supports the notion that this is a valid model for assessment of cognitive temporal lobe epilepsy comorbidities in preclinical research.

Viral Vector-Based Gene Therapy for Epilepsy: What Does the Future Hold?

In recent years, many pre-clinical studies have tested gene therapy approaches as possible treatments for epilepsy, following the idea that they may provide an alternative to conventional pharmacological and surgical options. Multiple gene therapy approaches have been developed, including those based on anti-sense oligonucleotides, RNA interference, and viral vectors. In this opinion article, we focus on translational issues related to viral vector-mediated gene therapy for epilepsy. Research has advanced dramatically in addressing issues like viral vector optimization, target identification, strategies of gene expression, editing or regulation, and safety. Some of these pre-clinically validated potential gene therapies are now being tested in clinical trials, in patients with genetic or focal forms of drug-resistant epilepsy. Here, we discuss the ongoing translational research and the advancements that are needed and expected in the near future. We then describe the clinical trials in the pipeline and the further challenges that will need to be addressed at the clinical and economic levels. Our optimistic view is that all these issues and challenges can be overcome, and that gene therapy approaches for epilepsy will soon become a clinical reality.

Sampling Cerebrospinal Fluid and Blood from Lateral Tail Vein in Rats during EEG Recordings.

Because the composition of body fluids reflects many physiological and pathological dynamics, biological liquid samples are commonly obtained in many experimental contexts to measure molecules of interest, such as hormones, growth factors, proteins, or small non-coding RNAs. A specific example is the sampling of biological liquids in the research of biomarkers for epilepsy. In these studies, it is desirable to compare the levels of molecules in cerebrospinal fluid (CSF) and in plasma, by withdrawing CSF and plasma in parallel and considering the time distance of the sampling from and to seizures. The combined CSF and plasma sampling, coupled with video-EEG monitoring in epileptic animals, is a promising approach for the validation of putative diagnostic and prognostic biomarkers. Here, a procedure of combined CSF withdrawal from cisterna magna and blood sampling from the lateral tail vein in epileptic rats that are continuously video-EEG monitored is described. This procedure offers significant advantages over other commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, and reduced time of anesthesia. Additionally, it can be used to obtain CSF and plasma samples in both tethered and telemetry EEG recorded rats, and it may be used repeatedly across multiple days of experiment. By minimizing the stress due to sampling by shortening isoflurane anesthesia, measures are expected to reflect more accurately the true levels of investigated molecules in biofluids. Depending on the availability of an appropriate analytical assay, this technique may be used to measure the levels of multiple, different molecules while performing EEG recording at the same time.

Metabotropic glutamate receptor subtype 8 in the amygdala modulates thermal threshold, neurotransmitter release, and rostral ventromedial medulla cell activity in inflammatory pain.

The amygdala is a crucial area in controlling the threshold of pain and its emotional component. The present study has evaluated the effect of a metabotropic glutamate 8 receptor (mGluR8) stimulation in the central nucleus of the amygdala (CeA) on the thermoceptive threshold and on CeA serotonin (5-HT), glutamate (Glu), and GABA release in normal and carrageenan-induced inflammatory pain conditions in rats. Furthermore, the activity of rostral ventromedial medulla (RVM) putative "pronociceptive" ON and "antinociceptive" OFF cells has been evaluated. (S)-3,4-Dicarboxyphenylglycine [(S)-3,4-DCPG], a selective mGluR8 agonist, administered into the CeA, did not change 5-HT, Glu, and GABA release, or the thermoceptive threshold, nor did it modify the activity of ON and OFF cells of the RVM in normal animals. In rats treated with carrageenan, intra-CeA (S)-3,4-DCPG perfusion produced antinociception, and increased 5-HT and Glu, whereas it decreased GABA release. Intra-CeA (S)-3,4-DCPG inhibited ON and increased OFF cell activities. Furthermore, an increase in mGluR8 gene, protein, and staining, the latter being associated with vesicular GABA transporter-positive profiles, has been found in the CeA after carrageenan-induced inflammatory pain. These results show that stimulation of mGluR8, which was overexpressed within the CeA in inflammatory pain conditions, inhibits nociceptive behavior. Such an effect is associated with an increase in 5-HT and Glu release, a decrease in GABA, and the inhibition of ON- and the stimulation of OFF-cell activities within RVM.

Loss of cortical GABA terminals in Unverricht-Lundborg disease.

Unverricht-Lundborg disease (ULD) is the most common progressive myoclonic epilepsy. Its etiology has been identified in a defect of a protease inhibitor, cystatin B (CSTB), but the mechanism(s) by which this defect translates in the clinical manifestations of the disease are still obscure. We tested the hypothesis that ULD is accompanied by a loss of cortical GABA inhibition in a murine model (the CSTB knockout mouse) and in a human case. Cortical GABA signaling has been investigated measuring VGAT immunohistochemistry (a histological marker of the density of GABA terminals), GABA release from synaptosomes and paired-pulse stimulation. In CSTB knockout mice, a progressive decrease in neocortex thickness was found, associated with a prevalent loss of GABA interneurons. A marked reduction in VGAT labeling was found in the cortex of both CSTB knockout mice and an ULD patient. This implicates a reduction in GABA synaptic transmission, which was confirmed in the mouse model as reduction in GABA release from isolated nerve terminals and as loss of electrophysiologically measured GABA inhibition. The alterations in VGAT immunolabeling progressed in time, paralleling the worsening of myoclonus. These results provide direct evidence that loss of cortical GABA input occurs in a relevant animal model and in a case of human ULD, leading to a condition of latent hyperexcitability that favors myoclonus and seizures. These findings contribute to the understanding of the pathogenic mechanism of ULD and of the neurobiological basis of the effect of currently employed drugs.

Synthetic cannabinoid JWH-073 alters both acute behavior and in vivo/vitro electrophysiological responses in mice.

JWH-073 is a synthetic cannabinoid (SCB) that is illegally marketed within an "herbal blend", causing psychoactive effects more intense than those produced by Cannabis. Users report that JWH-073 causes less harmful effects than other SCBs, misrepresenting it as a "safe JWH-018 alternative", which in turn prompts its recreational use. The present study is aimed to investigate the in vivo pharmacological activity on physiological and neurobehavioral parameters in male CD-1 mice after acute 1 mg/kg JWH-073 administration. To this aim we investigate its effect on sensorimotor (visual, acoustic, and tactile), motor (spontaneous motor activity and catalepsy), and memory functions (novel object recognition; NOR) in mice coupling behavioral and EEG data. Moreover, to clarify how memory function is affected by JWH-073, we performed in vitro electrophysiological studies in hippocampal preparations using a Long-Term Potentiation (LTP) stimulation paradigm. We demonstrated that acute administration of JWH-073 transiently decreased motor activity for up to 25 min and visual sensorimotor responses for up to 105 min, with the highest effects at 25 min (~48 and ~38%, respectively), while the memory function was altered up to 24 h (~33%) in treated-mice as compared to the vehicle. EEG in the somatosensory cortex showed a maximal decrease of α (~23%) and γ (~26%) bands at 15 min, ß (~26%) band at 25 min, a maximal increase of θ (~14%) band at 25 min and δ (~35%) band at 2 h, and a significant decrease of θ (~18%), α (~26%), and ß (~10%) bands during 24 h. On the other hand, EEG in the hippocampus showed a significant decrease of all bands from 10 min to 2 h, with the maximal effect at 30 min for θ (~34%) and γ (~26%) bands and 2 h for α (~36%), ß (~29%), and δ (~15%) bands. Notably, the δ band significant increase both at 5 min (~12%) and 24 h (~19%). Moreover, in vitro results support cognitive function impairment (~60% of decrease) by interfering with hippocampal synaptic transmission and LTP generation. Our results suggest that JWH-073 deeply alters brain electrical responsiveness with minor behavioral symptoms. Thus, it poses a subtle threat to consumers who mistakenly consider it safer than other SCBs.

Low-dose 7,8-Dihydroxyflavone Administration After Status Epilepticus Prevents Epilepsy Development.

Temporal lobe epilepsy often manifests months or even years after an initial epileptogenic insult (e.g., stroke, trauma, status epilepticus) and, therefore, may be preventable. However, no such preventive treatment is currently available. Aim of this study was to test an antioxidant agent, 7,8-dihydroxyflavone (7,8-DHF), that is well tolerated and effective in preclinical models of many neurological disorders, as an anti-epileptogenic drug. However, 7,8-DHF also acts as a TrkB receptor agonist and, based on the literature, this effect may imply an anti- or a pro-epileptogenic effect. We found that low- (5 mg/kg), but not high-dose 7,8-DHF (10 mg/kg) can exert strong anti-epileptogenic effects in the lithium-pilocarpine model (i.e., highly significant reduction in the frequency of spontaneous seizures and in the time to first seizure after status epilepticus). The mechanism of these different dose-related effects remains to be elucidated. Nonetheless, considering its excellent safety profile and antioxidant properties, as well as its putative effects on TrkB receptors, 7,8-DHF represents an interesting template for the development of effective and well-tolerated anti-epileptogenic drugs.

Anti-epileptogenic effect of NRP2945 in the pilocarpine model of temporal lobe epilepsy.

Innovative therapeutic strategies are highly needed to tackle the major medical needs of epilepsy, like prevention of epilepsy development in at-risk individuals, treatment of severe and drug-resistant forms, control of co-morbidities. The Neural Regeneration Peptide NRP2945 (a peptidomimetic analogue of the human CAPS-2 protein) has been recently found to exert many potentially anti-epileptic effects, for example increased neuronal survival and differentiation. In the present study, we tested the effects of NRP2945 on the development of epilepsy (epileptogenesis) and on chronic, spontaneous seizures, by using the pilocarpine model of temporal lobe epilepsy. We found that NRP2945 exerts a robust anti-epileptogenic effect, reducing the frequency of spontaneous seizures, exerting a significant neuroprotective effect and attenuating anxiety-like behaviors and cognitive impairment. These effects appear to depend on modulation of the epileptogenesis process and not on seizure suppression, because NRP2945 did not reduce frequency or duration of spontaneous seizures when administered to already epileptic animals. These findings may form the basis for a preventive therapy for individuals at-risk of developing epilepsy.

Improvement of HSV-1 based amplicon vectors for a safe and long-lasting gene therapy in non-replicating cells.

A key factor for developing gene therapy strategies for neurological disorders is the availability of suitable vectors. Currently, the most advanced are adeno-associated vectors that, while being safe and ensuring long-lasting transgene expression, have a very limited cargo capacity. In contrast, herpes simplex virus-based amplicon vectors can host huge amounts of foreign DNA, but concerns exist about their safety and ability to express transgenes long-term. We aimed at modulating and prolonging amplicon-induced transgene expression kinetics in vivo using different promoters and preventing transgene silencing. To pursue the latter, we deleted bacterial DNA sequences derived from vector construction and shielded the transgene cassette using AT-rich and insulator-like sequences (SAm technology). We employed luciferase and GFP as reporter genes. To determine transgene expression kinetics, we injected vectors in the hippocampus of mice that were longitudinally scanned for bioluminescence for 6 months. To evaluate safety, we analyzed multiple markers of damage and performed patch clamp electrophysiology experiments. All vectors proved safe, and we managed to modulate the duration of transgene expression, up to obtaining a stable, long-lasting expression using the SAm technology. Therefore, these amplicon vectors represent a flexible, efficient, and safe tool for gene delivery in the brain.

Effects of URB597, an inhibitor of fatty acid amide hydrolase (FAAH), on analgesic activity of paracetamol.

OBJECTIVES: Paracetamol is converted to an active metabolite AM404 via fatty acid amide hydrolase (FAAH). The aim of the present study was to ascertain whether a FAAH inhibitor URB597 antagonizes paracetamol analgesic activity (and to asses by this way the role of FAAH in analgesic activity of paracetamol). METHODS: The interaction between a FAAH inhibitor URB597 and paracetamol was investigated in the writhing test in mice using an isobolographic analysis. RESULTS: URB597 or paracetamol alone and in combinations produced dose-dependent antinociceptive effects. ED50 values were estimated for the individual drugs and an isobologram was constructed. The observed ED50 value for the URB57-paracetamol combination was 0.097 (0.062-0.247) mg/kg. This value did not differ significantly from the theoretical additive ED50 value for the URB597-paracetamol combination which was 0.108 (0.059-0.198) mg/kg. Thus, inhibition of FAAH by URB597 was not followed by the lack of analgesic activity in paracetamol. CONCLUSION: The present results suggest that the analgesic activity of paracetamol is not dependent solely on FAAH metabolic conversion to AM404 and that paracetamol exerts analgesic activity also by additional mechanisms.

Intraperiaqueductal gray glycine and D-serine exert dual effects on rostral ventromedial medulla ON- and OFF-cell activity and thermoceptive threshold in the rat.

We have studied the involvement of the N-methyl-D-aspartate receptor (NMDAR) glycine site and the strychnine-sensitive glycine receptor (GlyR) in the ventrolateral periaqueductal gray (VL-PAG) on nociceptive behavior (tail flick) and pain-related changes on neuronal activity in the rostral ventromedial medulla (RVM). Glycine or D-serine increased the tail-flick latency, reduced OFF-cell pause, and delayed its onset and increased the time between the onset of the OFF-cell pause and the tail withdrawal. Conversely, they decreased the ongoing activity of the ON cell, the tail-flick-induced ON-cell firing, whereas they delayed the onset of increased tail-flick-induced ON-cell firing. Also, glycine or D-serine reduced the interval between the onset of the increased ON-cell firing and tail withdrawal. Whereas 7-Cl-kynurenic acid (7-Cl-KYN) prevented such effects, strychnine did not do so. A higher dose of 7-Cl-KYN or strychnine was per se able to reduce or increase tail-flick latency and increase or reduce ON-cell activities, respectively. A higher dose of glycine was hyperalgesic in the presence of 7-Cl-KYN, whereas such an effect was prevented by strychnine. These data suggest 1) a dual role of glycine in producing hyperalgesia or analgesia by stimulating the GlyR or the NMDARs within the VL-PAG, respectively; 2) consistently that RVM ON and OFF cells display opposite firing patterns to the stimulation of the VL-PAG NMDAR glycine site and GlyR activation; and 3) a tonic role of these receptors within the VL-PAG-RVM antinociceptive descending pathway.

A determination of submicromolar concentrations of glycine in periaqueductal gray matter microdialyzates using capillary zone electrophoresis with contactless conductivity detection.

CE with contactless conductivity detection has been used to determine the glycine neurotransmitter in periaqueductal gray matter (PAG) of rats. The LOD for glycine has been decreased to a value of 0.2 microM by adding 75% v/v of ACN to the samples and increasing the sample zone introduced to a value of 20% of the overall capillary length. The repeatabilities of the analyte migration times and the zone areas amount to 2.1 and 2.7%, respectively. The optimized CE/contactless conductivity detection method makes it possible to determine the micromolar concentrations of glycine in PAG microdialyzates without the necessity of sample derivatization. It follows from a pharmacological study that a local inflammation initiated by an application of carrageenan increased the glycine concentration in the rat PAG seven times, compared with a control. The glycine level in PAG can be decreased and the pain suppressed by administering paracetamol.

Synergistic interaction between rilmenidine and ibuprofen in the writhing test in mice.

OBJECTIVES: The aim of the study was to ascertain whether rilmenidine, a second generation imidazoline-alpha-2-adrenoreceptor agonist, is able to increase analgesic effects of ibuprofen in the writhing test in mice. Experimental studies combining these agents have not yet been published. METHODS: An acetic acid (0.7%) solution was injected into the peritoneal cavity and the number of writhes was counted. The influence on locomotor performance was tested using the rotarod test. RESULTS: Rilmenidine, ibuprofen, and rilmenidine-ibuprofen fixed-ratio combinations produced dose-dependent antinociceptive effects. ED50 values were estimated for the individual drugs and an isobologram was constructed. The derived theoretical additive ED50 value for the rilmenidine-ibuprofen combination was 34.00 +/- 9.39 mg/kg. This value was significantly greater than the observed ED50 value which was 18.07 +/- 5.41 mg/kg, indicating a synergistic interaction. Rilmenidine did not impair motor coordination, as measured by the rotarod test, at antinociceptive and higher doses. CONCLUSIONS: The present results suggest that rilmenidine enhances the analgesic activity of ibuprofen. If rilmenidine produces antinociception in humans, then the synergistic antinociception of rilmenidine with ibuprofen could offer therapeutic advantage for clinical treatment of pain.

Gene Therapy Tools for Brain Diseases.

Neurological disorders affecting the central nervous system (CNS) are still incompletely understood. Many of these disorders lack a cure and are seeking more specific and effective treatments. In fact, in spite of advancements in knowledge of the CNS function, the treatment of neurological disorders with modern medical and surgical approaches remains difficult for many reasons, such as the complexity of the CNS, the limited regenerative capacity of the tissue, and the difficulty in conveying conventional drugs to the organ due to the blood-brain barrier. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. Gene therapy can result in a stable or inducible expression of transgene(s), and can allow a nearly specific expression in target cells. In this review, we will discuss the most commonly used tools for the delivery of genetic material in the CNS, including viral and non-viral vectors; their main applications; their advantages and disadvantages. We will discuss mechanisms of genetic regulation through cell-specific and inducible promoters, which allow to express gene products only in specific cells and to control their transcriptional activation. In addition, we will describe the applications to CNS diseases of post-transcriptional regulation systems (RNA interference); of systems allowing spatial or temporal control of expression [optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)]; and of gene editing technologies (CRISPR/Cas9, Zinc finger proteins). Particular attention will be reserved to viral vectors derived from herpes simplex type 1, a potential tool for the delivery and expression of multiple transgene cassettes simultaneously.

Sensitive chiral high-performance liquid chromatographic determination of anthelmintic flubendazole and its phase I metabolites in blood plasma using UV photodiode-array and fluorescence detection Application to pharmacokinetic studies in sheep.

Although benzimidazole anthelmintic flubendazole, methyl ester of [5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamic acid, is extensively used in veterinary and human medicine for the treatment of gastrointestinal parasitic helminth infections, reliable data about its pharmacokinetics in various species have not been reported. Our previous work [M. Nobilis, Th. Jira, M. Lísa, M. Holcapek, B. Szotáková, J. Lamka, L.Skálová, J. Chromatogr. A 1149 (2007) 112-120] had described the stereospecificity of carbonyl reduction during phase I metabolic experiments in vitro. For in vivo pharmacokinetic studies, further improvement and optimization of bioanalytical HPLC method in terms of sensitivity and selectivity was necessary. Hence, a modified chiral bioanalytical HPLC method involving both UV photodiode-array and fluorescence detection for the determination of flubendazole, both enantiomers of reduced flubendazole and hydrolyzed flubendazole in the extracts from plasma samples was tested and validated. Albendazole was used as an internal standard. Sample preparation process involved a pH-dependent extraction of the analytes from the blood plasma into tert-butylmethyl ether. Chromatographic separations were performed on a Chiralcel OD-R 250 mm x 4.6mm column with mobile phase methanol-1M NaClO(4) (75:25, v/v) at the flow rate 0.5 ml min(-1). In quantitation, selective UV absorption maxima of 290 nm (for reduced flubendazole), 295 nm (for albendazole), 310 nm (for flubendazole) and 330 nm (for hydrolyzed flubendazole) were used in the UV photodiode-array detection, and lambda(exc.)/lambda(emis.)=228 nm/310 nm (for reduced flubendazole) and lambda(exc.)/lambda(emis.)=236 nm/346 nm (for albendazole) were set on the fluorescence detector. The fluorescence detection was approximately 10-times more sensitive than the UV detection. Each HPLC run lasted 27 min. The validated chiral HPLC-PDA-FL method was employed in the pharmacokinetic studies of flubendazole in sheep. The stereospecificity of the enzymatic carbonyl reduction of flubendazole was also observed in vivo. (+)-Reduced flubendazole was found to be the principal metabolite in ovine blood plasma and only low concentrations of hydrolyzed flubendazole, the parent flubendazole and (-)-reduced flubendazole were detected in this biomatrix.

Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.

Microdialysis is a well-established neuroscience technique that correlates the changes of neurologically active substances diffusing into the brain interstitial space with the behavior and/or with the specific outcome of a pathology (e.g., seizures for epilepsy). When studying epilepsy, the microdialysis technique is often combined with short-term or even long-term video-electroencephalography (EEG) monitoring to assess spontaneous seizure frequency, severity, progression and clustering. The combined microdialysis-EEG is based on the use of several methods and instruments. Here, we performed in vivo microdialysis and continuous video-EEG recording to monitor glutamate and aspartate outflow over time, in different phases of the natural history of epilepsy in a rat model. This combined approach allows the pairing of changes in the neurotransmitter release with specific stages of the disease development and progression. The amino acid concentration in the dialysate was determined by liquid chromatography. Here, we describe the methods and outline the principal precautionary measures one should take during in vivo microdialysis-EEG, with particular attention to the stereotaxic surgery, basal and high potassium stimulation during microdialysis, depth electrode EEG recording and high-performance liquid chromatography analysis of aspartate and glutamate in the dialysate. This approach may be adapted to test a variety of drug or disease induced changes of the physiological concentrations of aspartate and glutamate in the brain. Depending on the availability of an appropriate analytical assay, it may be further used to test different soluble molecules when employing EEG recording at the same time.