Toward evidence-based severity assessment in mouse models with repeated seizures: I. Electrical kindling.

OBJECTIVE: Ethical decisions about an allowance for animal experiments need to be based on scientifically sound information about the burden and distress associated with the experimental procedure and models. Thereby, species differences need to be considered for recommendations regarding evidence-based severity assessment and refinement measures. METHODS: A comprehensive analysis of behavioral patterns and corticosterone or its metabolites in serum and feces was completed in kindled mice. The impact of kindling via two different stimulation sites in the amygdala and hippocampus was determined. Data were compared to those from naive and electrode-implanted groups. RESULTS: Amygdala and hippocampus kindled mice exhibited comparable behavioral patterns with increased activity in the open field, reduced anxiety-associated behavior in the elevated-plus maze, and increased anhedonia-associated behavior in the saccharin preference test. In addition, repeated stimulation of the hippocampus caused a reduction in burrowing behavior and an increase in active social interaction. Levels of corticosterone and its metabolites were not altered in serum or feces, respectively. A comparison of mouse data with findings from amygdala kindled rats confirmed pronounced species differences in behavioral patterns associated with the kindling process. SIGNIFICANCE: Taken together the findings suggest a severity classification for the mouse kindling paradigms as moderate regardless of the stimulation site. The outcome of the species comparison provides valuable guidance for species selection for studies exploring behavioral comorbidities. In this context, it is emphasized that the mouse kindling paradigms seem to be well suited for studies exploring the link between ictal events and network alterations on the one hand, and hyperactivity and anhedonia-associated behavior on the other hand. Moreover, the underlying pathophysiological mechanisms and the impact of therapeutic interventions on these behavioral alterations can be studied in these paradigms providing guidance for the clinical management of respective psychiatric comorbidities in patients.

Design of composite measure schemes for comparative severity assessment in animal-based neuroscience research: A case study focussed on rat epilepsy models.

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.

Toward evidence-based severity assessment in rat models with repeated seizures: I. Electrical kindling.

OBJECTIVE: Rodent epilepsy models can significantly contribute to our understanding of pathophysiological mechanisms and to validation of biomarker and target candidates. Evidence-based severity assessment is a presupposition for the ethical evaluation of animal experimentation allowances as well as for the development of efficacious refinement concepts. METHODS: Aiming to improve our understanding of the impact of experimental procedures and repeated seizures, we have completed a comprehensive behavioral and biochemical analysis assessing various parameters that can inform about the influence of an electrical kindling paradigm on well-being in rats. Thereby, we have focused on the immediate effects of phases with focal and generalized seizures with behavioral testing during kindling acquisition. RESULTS: Electrode implantation exerted mild effects on anxiety-associated behavior and reduced serum corticosterone at 3 weeks, but not 7 weeks, following surgery. Analysis in kindled rats excluded any relevant impact of focal seizures on behavioral and biochemical parameters. Assessment in rats with generalized seizures revealed an impact on nest complexity scores, nest soiling, and selected parameters in paradigms evaluating anxiety-associated behavior. Moreover, serum corticosterone levels, but neither hair corticosterone nor fecal corticosterone metabolite concentrations were lowered as a consequence of repeated generalized seizures. The assessment of various other behavioral and biochemical parameters did not reveal any other relevant effects of generalized seizures. Cross-correlation analysis suggested that assessment of nest building and maintenance can provide information comparable to that from more elaborate behavioral assays. This finding provides first evidence that nest scoring might serve as a simple and valid approach to evaluate rat well-being during routine assessment schemes. SIGNIFICANCE: The findings argue against a persistent level of pronounced distress and suggest a classification of the kindling paradigm as a model with moderate severity based on a longer-lasting mild impact on animal behavioral patterns. This suggestion provides a basis for a prospective and retrospective case-by-case severity assessment.

Assessment of luteolin isolated from Eclipta alba leaves in animal models of epilepsy.

CONTEXT: Eclipta alba (Linn) Hassk. (Asteraceae) has been reported to be a nerve tonic and has been used to treat epilepsy in folk medicine. OBJECTIVE: The present study isolates and characterizes luteolin from E. alba and evaluates its antiepileptic potential in chemically induced acute and chronic models in mice. MATERIALS AND METHODS: The methanol extract (16.85% w/w) of E. alba leaves was subjected to fractionation for isolation of luteolin. In acute pentylenetetrazole (PTZ) model, luteolin (5, 10, 20 mg/kg, i.p.) was administered 30 min prior to PTZ injection (100 mg/kg) in Swiss albino mice. Kindling was induced by chronic administration of PTZ (35 mg/kg) on every alternate day (48 days). Luteolin was investigated on the course of kindling development and oxidative stress markers [reduced glutathione (GSH) and malondialdehyde (MDA)] in kindled mice. RESULTS: Single-dose pretreatment with luteolin (10 and 20 mg/kg, i.p.) was found to be effective in an acute PTZ model (100% protection from mortality) and it did not exhibit any effect on motor coordination at the same doses. PTZ-induced kindling was significantly (p < 0.001) prevented by luteolin (5, 10, 20 mg/kg, i.p.) in a dose-dependent manner. Luteolin restored levels of reduced GSH (p < 0.001) and decreased the level of MDA (p < 0.001), a marker of lipid peroxidation. DISCUSSION AND CONCLUSION: The results of the present study demonstrated that luteolin had an anticonvulsant effect in an acute PTZ model. Luteolin exhibited and inhibitory effect on the course of kindling and associated oxidative stress and hence could be a potential molecule in the treatment of epilepsy.

Ahogamiento incompleto, valoración de la injuria cerebral al ingreso: terapia intensiva pediátrica / Nearly drowning, valuation of the cerebral injury when entering the hospital: intensive pediatric therapy

Se realizó una investigación descriptiva retrospectiva que incluyó a todos los pacientes que fueron ingresados en el Servicio de Terapia Intensiva del Hospital Eliseo Noel Caamaño, en el período comprendido desde junio de 1982 hasta junio de 2004, con el diagnóstico de Ahogamiento Incompleto, con el propósito de determinar el estado neurológico y su relación con diferentes variables que pueden haber influido en la intensidad del daño, así como la sobrevivencia final de los mismos. Los resultados obtenidos nos permitieron conocer el manejo del paciente pediátrico que ha sufrido este tipo de accidentes, así como su estado a la llegada al Servicio. El 68 por ciento de los pacientes tuvieron una afección de moderada a severa según las escalas de Conn y Glasgow, además existió una correlación de hasta un 97 por ciento entre dichos métodos de evaluación. Dentro de las variables que influenciaron en el estado de los niños al ingreso se destacó un tiempo de inmersión mayor de 5 minutos para los clasificados como severos, 70 por ciento de la muestra, fallecieron el 7 por ciento de todos los pacientes estudiados, todos ellos clasificados como severos.

We carried out a descriptive retrospective study including all the patients that were entered in the Service of Intensive Therapy of the hospital Eliseo Noel Caamaño, in the period from June 1982 to June 2004, with the diagnosis of nearly drowning, with the purpose of determining the neurological state and its relation with different variables that might have influence in the intensity of the damage, as well as in the final surviving of the patients. The obtained results allowed us knowing the managing of the paediatrics patient that have suffered this kind of accidents, as well as their state at the arrival to the service; 68 percent of the patients had a moderated to severe affection according to the Conn and Glasgow scales; moreover, there was a correlation of up to 97 percent between these evaluation methods. Among the variables that influenced in the state of the children at the entering, there was an immersion time of more than 5 minutes for those classified as severe, 70 percent of the sample; 7 percent of all the studied patients died, all of them classified as severe.

Assessment of anxiety-like behaviors in female rats bred for differences in kindling susceptibility and amygdala excitability.

Two rat lines bred for kindling susceptibility were previously observed to engage in different behavioral strategies in tests of emotionality. In order to extend past research on defensive behaviors in these strains which largely used males, Fast- and Slow-kindling females were assessed for anxiety-like behaviors in a number of aversive paradigms. Fast rats entered and spent more time in the open arms and spent less time in the closed arms of the elevated plus-maze (EPM) compared to Slow animals. Fast rats had higher conditioned suppression ratios across testing days, defecated less often during conditioning, and successfully disinhibited suppression during extinction in the conditioned emotional response (CER) paradigm compared to Slow-kindlers. In order to pursue these differences in emotional reactivity between the strains and differentiate negative affect from motivational, learning, and impulsive explanations, a separate group of animals were assessed in the light-enhanced acoustic startle chamber, a test of anxiety. When initially exposed to a bright-light, Slow rats significantly increased their startle response while this was not observed in the Fast strain. In combination with previous research on these strains, the present data tentatively suggest that Fast and Slow animals utilize different neural systems in tests of fear and anxiety which may have been co-selected with the direct selection of amygdala-kindling susceptibility.

The price of seizure control: dynorphins in interictal and postictal psychosis.

Postictal and interictal psychoses are relatively common complicating factors in the clinical course of epilepsy, yet their neurobiological substrates are poorly understood. Recent evidence shows that kappa opioid receptor (KOR) activation elicits anticonvulsant and psychotomimetic effects. In view of this background, here we introduce the hypothesis that epilepsy-related psychoses may partially result from excessive hippocampal dynorphin release and kappa opioid receptor overstimulation aimed at seizure control.

A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.

Safety of intraoperative transcranial electrical stimulation motor evoked potential monitoring.

This article reviews intraoperative transcranial electrical stimulation (TES) motor evoked potential (MEP) monitoring safety based on comparison with other clinical and experimental brain stimulation methods and clinical experience in more than 15000 cases. Comparative analysis indicates that brain damage and kindling are highly unlikely. There have been remarkably few adverse events. Pulse train TES-induced or coincidental seizures (n = 5) are rare, probably because of very brief (<0.03 second) stimuli, anesthesia, and the general absence of predisposing cerebral conditions. Soft bite blocks may prevent tongue or lip laceration (n = 29) or mandibular fracture (n = 1). Rare cardiac arrhythmia (n = 5) and intraoperative awareness (n = 1) may be coincidental. Minor scalp burns (n = 2) are rare. Although possible, no spinal epidural recording electrode complications or injuries resulting from TES-induced movement were found. There have been no recognized adverse neuropsychological effects, headaches, or endocrine disturbances. Comprehensive relative contraindications include epilepsy, cortical lesions, convexity skull defects, raised intracranial pressure, cardiac disease, proconvulsant medications or anesthetics, intracranial electrodes, vascular clips or shunts, and cardiac pacemakers or other implanted biomedical devices. Otherwise unexplained intraoperative seizures and possibly arrhythmias are indications to abort TES. With appropriate precautions in expert hands, the well-established benefits of TES MEP monitoring decidedly outweigh the associated risks.

Assessment of the role of kindling in the pathogenesis of alcohol withdrawal seizures and delirium tremens.

The aim of this study was to evaluate the hypothetical role of kindling phenomenon in the development and course of alcohol withdrawal (AW) seizures and delirium tremens (DT). The 2186 medical records of 1179 patients hospitalized in Nowowiejski Hospital in Warsaw from 1973 to 1987 were reviewed using a structured questionnaire. Investigating the role of kindling, a course of consecutive AW episodes of patients hospitalized several times was analyzed. The relationships of withdrawal seizures with the duration of alcohol abuse, the number of prior detoxification episodes, and other variables were also studied. Increasing severity of AW symptoms was observed during the course of consecutive episodes in 22.5% of patients. The first episode of DT was preceded by withdrawal seizures in 11% of cases. First-ever withdrawal seizures occurred more frequently in patients with head injury in the past and with coexisting symptoms of alcohol liver disease. Occurrence of withdrawal seizures and DTs did not correlate with the number of previous withdrawal episodes or with the length of period of intensive drinking. We concluded that the kindling model could be applied only to some cases in the development of AW seizures and DTs. Kindling should be considered as one of the multiple mechanisms involved in the pathogenesis of AW delirium.

Pharmacogenetic assessment of the effects of carbamazepine on cocaine-kindled and cocaine-induced seizures.

The effects of chronic carbamazepine (CBZ) on the development and expression of cocaine-kindled seizures and seizures produced by an acute injection of cocaine were evaluated in BALB/cByJ, C57Bl/6J and SJL/J mice. The repeated administration of a subconvulsant dose of cocaine initially resulted in the development of an increased sensitivity to the convulsant effects of cocaine in the three strains. Chronic, dietary carbamazepine attenuated this initial sensitization to cocaine-induced seizures. While the continued administration of cocaine resulted in a relatively permanent sensitization to cocaine-induced seizures among SJL mice, tolerance to cocaine-induced seizures ultimately developed among C57 mice and to a lesser degree among BALB mice. Genetic factors were found to mediate the effects of chronic CBZ on the development of sensitization and/or tolerance to the convulsant effects of cocaine. Among BALB mice, chronic CBZ appears to have eliminated the development of tolerance to cocaine-induced seizures and allowed an underlying sensitization to be manifest. Among SJL mice, however, the sensitization observed following repeated cocaine injections was reduced, but not eliminated. Genetic factors were also found to be associated with the effects of CBZ on seizures induced by the acute administration of cocaine. BALB and C57 mice, but not SJL mice, chronically treated with dietary CBZ were less susceptible to a consulvant dose of cocaine than their corresponding dietary controls for at least 72 h after stopping CBZ administration. In addition, there were genotype-specific lethal effects associated with the concurrent administration of CBZ and cocaine.

The effect of interstimulation interval on the assessment of anticonvulsant drug potency in fully kindled rats.

Multiple stimulation regimes with interstimulation intervals of 0.25-2 h were investigated in fully kindled rats as methods of testing the time course and potency of anticonvulsant drugs after a single administration. Protocols with conventional interstimulation intervals of 1-3 days were used for comparison. Prior to the drug experiments, the different stimulation regimes were examined in drug-naive, kindled rats in order to determine the extent of postseizure inhibition occurring with such protocols. All stimulations were carried out with a suprathreshold current of 500 microA. Using protocols with 3-9 stimulations within 8 h, seizure severity was relatively stable, but motor seizure duration was reduced in most experiments. Both decreases and increases were observed with respect to afterdischarge duration (ADD). The increases in ADD were primarily due to the appearance of 'secondary' afterdischarges with small amplitude, which were associated with immobility, intermittent facial clonus and head nodding. After a series of stimulations at short intervals, reduced seizure severity was observed after this series for at least 1 week, so that an interval of at least 2 weeks had to be interposed between multistimulation experiments in the same group of rats. When the effects of carbamazepine, 15 mg/kg, were determined with 4 different stimulation regimes, it was found that the anticonvulsant potency of the drug was higher in experiments with short interstimulation intervals compared to conventional protocols with interstimulation intervals of 1-3 days, indicating synergistic effects between the drug and postictal inhibition. Indication for such synergism was also found when the animals were only stimulated once daily during the drug experiments. With higher doses of carbamazepine or phenobarbital, 30 mg/kg, the difference between the stimulation protocols was less marked. Furthermore, the time course of anticonvulsant action determined with different stimulation regimes was similar. The data indicate that multistimulation regimes can be used in kindled rats to determine the time course following single administration of anticonvulsant drugs, but such protocols may lead to an overestimation of anticonvulsant potency.

Antiepileptic drug development program: a cooperative effort of government and industry.

The most important step in antiepileptic drug discovery is the choice of an appropriate animal model for the initial screening as well as for the more complex procedures that elucidate mechanisms of action. The currently available models fall short in their inability to identify all drugs for all types of seizures in a mechanism-independent manner. Nevertheless, spontaneous models of epilepsy are the most commonly used, and chemically or electrically induced seizures in rodents can also identify potential anticonvulsants. In the latter models, the intensity of the seizure stimulus is of paramount importance. The Antiepileptic Drug Development Program evaluates approximately 800 compounds each year, using two models for preliminary screening. One model assesses the ability of a compound to prevent seizure spread; the other weighs the ability to raise seizure threshold. In vivo tests, featuring amygdala- and corneal-kindled seizures, and in vitro assays, employing gamma-aminobutyric acid (GABA) receptors and synaptosomal uptake of adenosine, define drug-drug interactions and elucidate the pharmacological profiles of potential anticonvulsants.

Physiological methods for assessment of Trimethyltin exposure.

Trimethyltin has been reported to produce morphological alterations in the brain which are primarily restricted to the limbic system. A variety of physiological measures of limbic system integrity are discussed in terms of their ability to detect TMT-induced dysfunction. In addition, several measures of sensory dysfunction are discussed. It is concluded that limbic system dysfunction induced by this compound is detected more efficiently by intrahippocampal evoked potentials than by more gross measures of dysfunction. It is also concluded that relying upon preliminary descriptions of pathological alterations to direct physiological studies may provide an incomplete description of neurotoxicity.

The effect of interstimulation interval on the assessment and stability of kindled seizure thresholds.

Two experiments were conducted to investigate the importance of the interstimulus interval (ISI) in the assessment of kindled seizure thresholds using an ascending method of limits. A third experiment examined the effect of varying the interval between successive threshold tests on threshold stability. No differences in afterdischarge (AD) or motor seizure (MS) threshold were observed when the ISI was either five minutes or 48 hours. However, a 30 second ISI yielded significantly higher AD and MS thresholds and shorter seizure duration compared to 1, 3, or 5 minute ISI's. AD and MS thresholds were found to be relatively stable over five successive tests when the inter-test interval was 48 hours. An inter-test interval of 24 hours, however, yielded progressively higher thresholds over the five tests.