Comparative severity assessment of animal models and experimental interventions is of utmost relevance for harm-benefit analysis during ethical evaluation, an animal welfare-based model prioritization as well as the validation of refinement measures. Unfortunately, there is a lack of evidence-based approaches to grade an animal\'s burden in a sensitive, robust, precise, and objective manner. Particular challenges need to be considered in the context of animal-based neuroscientific research because models of neurological disorders can be characterized by relevant changes in the affective state of an animal. Here, we report about an approach for parameter selection and development of a composite measure scheme designed for precise analysis of the distress of animals in a specific model category. Data sets from the analysis of several behavioral and biochemical parameters in three different epilepsy models were subjected to a principal component analysis to select the most informative parameters. The top-ranking parameters included burrowing, open field locomotion, social interaction, and saccharin preference. These were combined to create a composite measure scheme (CMS). CMS data were subjected to cluster analysis enabling the allocation of severity levels to individual animals. The results provided information for a direct comparison between models indicating a comparable severity of the electrical and chemical post-status epilepticus models, and a lower severity of the kindling model. The new CMS can be directly applied for comparison of other rat models with seizure activity or for assessment of novel refinement approaches in the respective research field. The respective online tool for direct application of the CMS or for creating a new CMS based on other parameters from different models is available at https://github.com/mytalbot/cms. However, the robustness and generalizability needs to be further assessed in future studies. More importantly, our concept of parameter selection can serve as a practice example providing the basis for comparable approaches applicable to the development and validation of CMS for all kinds of disease models or interventions.

BACKGROUND: Ocimum sanctum L. commonly known as tulsi (synonym of Ocimum tenuiflorum L.) is widely used in Ayurveda medicine and is having multitude neuromodulatory effect including the anticonvulsant effect in acute seizure models as per previous studies. In India, it is used for the treatment of epilepsy as traditional medicine. However, its role in chronic seizure model and interaction with newer antiepileptic drugs has not been investigated, which will enhance its translational value.
OBJECTIVE: Current study investigated the effect of Ocimum on chronic seizure model and its interaction with levetiracetam (LEV), a newer antiepileptic drug.
METHODS: The adjuvant role of Ocimum sanctum hydroalcoholic extracts (OSHE) 1000 mg/kg along with LEV 300 mg/kg was studied in adult male Wistar rats with mean weight of 227.84 ± 21.68 g using pentylenetetrazole (30 mg/kg, i.p.) kindling (K) (with maximum 24 injections on alternate days and challenge on 7th-day). Along with seizure score, neurobehavioral, brain tissue oxidative stress and histopathology status were assessed. Pharmacokinetic interaction was assessed between LEV and OSHE after 14 days of drug treatment.
RESULTS: K-LEV + OSHE had least seizure score during kindling and on the pentylenetetrazole-challenge test (p=0.031) than other kindling groups. Seizure protection was more in K-LEV + OSHE (85.72%) than others (K-LEV-42.86%, K-OSHE-42.86%, and K-Control-28.58%). Ocimum treated groups had better memory retention potential as evident from Morris water maze (MWM), passive avoidance test but not in an elevated plus maze test. Oxidative-stress was lower in Ocimum treated groups than K-Control group. As per histopathology, K-LEV + OSHE group had the least neuronal degeneration among kindling groups. There was no significant pharmacokinetic interaction between LEV and OSHE, except increased Tmax in LEV + OSHE group than LEV alone (p=0.009).
CONCLUSIONS: Ocimum per se and combination with levetiracetam treatment exerted better seizure control, memory retention, oxidative stress reduction, and neuronal structure preservation than kindling control group. There was a very minimal drug interaction between Ocimum and LEV. So, Ocimum as an adjuvant to LEV may be shelpful in enhancing the antiepileptic effect and also in minimizing the adverse effects.

Epilepsy affects about 1-2 % of world population as a chronic neurological disease that is manifested by repeated and consecutive seizures (Grone and Baraban, Nat Neurosci 18(3):339-343, 2015). There is no definitive therapy for epilepsy and antiepileptic drugs cannot offer a permanent and definitive cure for epilepsy, and most epileptic patients become drug resistant (Sasa, J Pharmacol Sci 100(5):487-494, 2006). Surgery and removal of the epileptic focus is a substitute method for treating drug-resistant patients and epilepsy surgery of either side of the brain improves seizure control. Temporal lobectomy is the most common epilepsy surgery and is associated with high success rates. Other studies have reported higher success rates for carefully selected temporal lobe seizure patients. Some physicians still consider temporal lobectomy an extreme procedure, citing the risks of side effects, including loss of memory, visual disturbances, and emotional change, associated with the removal of brain tissue (Spencer, Lancet Neurol 1(6):375-382, 2002; Wiebe et al., N Engl J Med 345(5):311-318, 2001; Yasargil et al., J Neurosurg 112(1):168-185, 2010). Nowadays, direct electrical stimulation (in the form of low- or high-frequency stimulation) in the location involved in seizures is used as a potentially suitable treatment method for this destructive disease in both laboratory animals and humans (Goodman et al., Epilepsia 46(1):1-7, 2005; Richardson et al., Epilepsia 44(6):768-777, 2003; Velasco et al., Epilepsia 41(2):158-169, 2000). Low-frequency stimulation causes less damage to the epileptic area and surrounding neuronal structures compared to high-frequency stimulation, and it can be a suitable option for patients suffering from epilepsy (Goodman et al., Epilepsia 46(1):1-7, 2005). Since the cellular mechanism of this stimulation is not clearly known, the purpose of this review research was to investigate the anticonvulsive effects of low-frequency electrical stimulation and the probable cellular mechanism involved in it.

Our previous studies carried out on the pilocarpine model of seizures showed that highly resolved elemental analysis might be very helpful in the investigation of processes involved in the pathogenesis of epilepsy, such as excitotoxicity or mossy fiber sprouting. In this study, the changes in elemental composition that occurred in the hippocampal formation in the electrical kindling model of seizures were examined to determine the mechanisms responsible for the phenomenon of kindling and spontaneous seizure activity that may occur in this animal model. X-ray fluorescence microscopy was applied for topographic and quantitative analysis of selected elements in tissues taken from rats subjected to repetitive transauricular electroshocks (ES) and controls (N). The detailed comparisons were carried out for sectors 1 and 3 of the Ammon\'s horn (CA1 and CA3, respectively), the dentate gyrus (DG) and hilus of DG. The obtained results showed only one statistically significant difference between ES and N groups, namely a higher level of Fe was noticed in CA3 region in the kindled animals. However, further analysis of correlations between the elemental levels and quantitative parameters describing electroshock-induced tonic and clonic seizures showed that the areal densities of some elements (Ca, Cu, Zn) strongly depended on the progress of kindling process. The areal density of Cu in CA1 decreased with the cumulative (totaled over 21 stimulation days) intensity and duration of electroshock-induced tonic seizures while Zn level in the hilus of DG was positively correlated with the duration and intensity of both tonic and clonic seizures.

A series of twelve compounds (Compounds RNH1-RNH12) of acid hydrazones of pyridine-3-carbohydrazide or nicotinic acid hydrazide was synthesized and evaluated for anticonvulsant activity by MES, scPTZ, minimal clonic seizure and corneal kindling seizure test. Neurotoxicity was also determined for these compounds by rotarod test. Results showed that halogen substitution at meta and para position of phenyl ring exhibited better protection than ortho substitution. Compounds RNH4 and RNH12, were found to be the active analogs displaying 6Hz ED50 of 75.4 and 14.77 mg/kg while the corresponding MES ED50 values were 113.4 and 29.3 mg/kg respectively. In addition, compound RNH12 also showed scPTZ ED50 of 54.2 mg/kg. In the series, compound RNH12 with trifluoromethoxy substituted phenyl ring was the most potent analog exhibiting protection in all four animal models of epilepsy. Molecular docking study has also shown significant binding interactions of these two compounds with 1OHV, 2A1H and 1PBQ receptors. Thus, N-[(meta or para halogen substituted) benzylidene] pyridine-3-carbohydrazides could be used as lead compounds in anticonvulsant drug design and discovery.

It has been shown that low-frequency stimulation (LFS) can induce anticonvulsant effects. In this study, the effect of different LFS frequencies on kindling induced behavioral and ultrastructural changes was investigated. For induction of kindled seizures in rats, stimulating and recording electrodes were implanted in perforant path and dentate gyrus, respectively. Animals were stimulated in a rapid kindling manner. Different groups of animals received LFS at different frequencies (0.5, 1 and 5 Hz) following kindling stimulations and their effects on kindling rate were determined using behavioral and ultrastructural studies. Kindling stimulations were applied for 7 days. Then, the animals were sacrificed and their dentate gyrus was sampled for ultrastructural studies under electron microscopy. All three used LFS frequencies (0.5, 1 and 5 Hz) had a significant inhibitory effect on kindling rate and decreased afterdischarge duration and the number of stimulations to achieve stage 4 and 5 seizures significantly. In addition, application of LFS prevented the increase in the post-synaptic density and induction of concave synaptic vesicles following kindling. There was no significant change between anticonvulsant effects of LFS at different frequencies. Obtained results show that LFS application can prevent the neuronal hyperexcitability by preventing the ultrastructural changes during kindling and this may be one of the mechanisms of LFS anticonvulsant effects.

As a continuation of our efforts to develop the azolylchromanone derivatives as potential anticonvulsant agents, we explored (Z)- and (E)-oxime ether derivatives of imidazolylchromanones bearing different lipophilic O-benzyl groups and tested their anticonvulsant activities in PTZ-kindling model of epilepsy. O-(2,4-Dichlorobenzyl) oximes 8a, 16a and 20a were significantly effective in delaying the onset of the PTZ-evoked seizures at the dose of 30mg/kg in kindled animals. The most effective compounds in delaying seizures were 7-chlorochromanone-O-(2,4-dichlorobenzyl) oximes 8a and 20a. SAR studies showed that introduction of a chlorine atom to the 7-position and/or a methyl group to the 2-position of the chroman ring resulted in an improvement of anti-seizure efficacy in O-(2,4-dichlorobenzyl) oxime series.

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OBJECTIVE: To examine antiepileptogenic and antiictogenic potential of retigabine (RTG) under conditions of rapid kindling epileptogenesis during different stages of development.
METHODS: The experiments were performed in postnatal day 14 (P14), P21, and P35 male Wistar rats. After stereotaxic implantation of hippocampal stimulating and recording electrodes, the effects of RTG on baseline afterdischarge (AD) properties were studied. Next, the animals underwent rapid kindling (sixty 10 s trains, bipolar 20 Hz square wave pulses delivered every 5 min). The progression of seizures (kindling acquisition), and responses to test stimulations after kindling (retention) were compared between RTG and vehicle-treated rats. Additionally, the effects of RTG on the severity of seizures in previously kindled animals were examined.
RESULTS: When administered intraperitoneally in doses that induced only mild, or no motor deficits, RTG significantly dampened brain excitability, evident as the increase of AD threshold and shortening of AD duration. During kindling, RTG delayed the development of focal seizures in P14 rats, and prevented the occurrence of full limbic seizures at all three ages. At P14 and P21, but not at P35, pretreatment with RTG prevented the establishment of kindling-induced enhanced seizure susceptibility. Administration of RTG to kindled animals decreased the severity of seizures induced by test stimulation. The effect was most prominent at P14.
CONCLUSIONS: RTG exerted both antiepileptogenic and antiictogenic effects under conditions of rapid kindling model. These effects were apparent during postneonatal, early childhood, and adolescent stages of development.

Kindling and impaired electroencephalophysiology have been suggested to play a role in the pathophysiology of premenstrual dysphoric disorder (PMDD). Levetiracetam is a novel antiepileptic drug which has shown strong anti-kindling activity in animal models of epilepsy. In this preliminary prospective study we examined the safety and efficacy of levetiracetam for the treatment of PMDD. One hundred twenty-three potential patients were prospectively screened to enroll seven patients into the open-label treatment phase of the study. PMDD was diagnosed per DSM-IV-TR criteria and two consecutive months of prospective ratings of Daily Record of Severity of Problems (DRSP). The Mini International Neuropsychiatric Interview (MINI) was used to exclude any co-morbid conditions. Levetiracetam was started at 250 mg qhs at the end of the first week of the follicular phase. Dosage was gradually increased up to 1,500 mg bid as tolerated or clinically effective. The treatment phase lasted 4 months. Response to treatment was evaluated by Clinical Global Impression (CGI) and DRSP scores. Six out of seven patients experienced a considerable decrease in their DRSP scores with levetiracetam, starting from the first treatment cycle. One patient dropped out of the study due to lack of efficacy after one cycle. Medication was fairly well tolerated. Improvements in food cravings and premenstrual headaches were also noted as unexpected benefits. Anticonvulsant medications, specifically levetiracetam, could be effective in the treatment of PMDD. Future double-blind, placebo controlled, randomized studies are warranted and should include larger number of patients.