An exploratory approach to identify microRNAs as circulatory biomarker candidates for epilepsy-associated psychiatric comorbidities in an electrical post-status epilepticus model.

Patients with epilepsy have a high risk of developing psychiatric comorbidities, and there is a particular need for early detection of these comorbidities. Here, in an exploratory, hypothesis-generating approach, we aimed to identify microRNAs as potential circulatory biomarkers for epilepsy-associated psychiatric comorbidities across different rat models of epilepsy. The identification of distress-associated biomarkers can also contribute to animal welfare assessment. MicroRNA expression profiles were analyzed in blood samples from the electrical post-status epilepticus (SE) model. Preselected microRNAs were correlated with behavioral and biochemical parameters in the electrical post-SE model, followed by quantitative real-time PCR validation in three additional well-described rat models of epilepsy. Six microRNAs (miR-376a, miR-429, miR-494, miR-697, miR-763, miR-1903) were identified showing a positive correlation with weight gain in the early post-insult phase as well as a negative correlation with social interaction, saccharin preference, and plasma BDNF. Real-time PCR validation confirmed miR-203, miR-429, and miR-712 as differentially expressed with miR-429 being upregulated across epilepsy models. While readouts from the electrical post-SE model suggest different microRNA candidates for psychiatric comorbidities, cross-model analysis argues against generalizability across models. Thus, further research is necessary to compare the predictive validity of rodent epilepsy models for detection and management of psychiatric comorbidities.

Correlation of Metabolic Syndrome with Redox Homeostasis Biomarkers: Evidence from High-Fat Diet Model in Wistar Rats.

Metabolic Syndrome (MetS) is an extremely complex disease. A non-balanced diet such as high-fat diet (HFD) induces metabolic dysfunction that could modify redox homeostasis. We here aimed at exploring redox homeostasis in male Wistar rats, following 8 weeks of HFD, correlating the eventual modification of selected biomarkers that could be associated with the clinical manifestations of MetS. Therefore, we selected parameters relative to both the glucose tolerance and lipid altered metabolism, but also oxidative pattern. We assessed some biomarkers of oxidative stress i.e., thiols balance, lipid peroxidation and antioxidant barriers, via the use of specific biochemical assays, individuating eventual cross correlation with parameters relative to MetS through a Principal Component Analysis (PCA). The present study shows that 8 weeks of HFD induce MetS in rats, altering glucose and lipid homeostasis and increasing visceral adipose tissue, but also impairing the physiological antioxidant responses that could not counteract the oxidative stress condition. Crucially, cross-correlation analysis suggested that the assessment of specific oxidative stress parameters reported here can provide information comparable to the more widely acquired biomarkers of Mets such as glucose tolerance. Lastly, hepatic steatosis in association with the oxidative stress condition was also highlighted by histological analysis. This research will elucidate the fundamental impact of these oxidative stress parameters on MetS induced in the HFD rat model, tracing paths for developing prevention approaches.

Metabolomic signature of the Dravet syndrome: A genetic mouse model study.

OBJECTIVE: Alterations in metabolic homeostasis can contribute to neuronal hyperexcitability and seizure susceptibility. Although the pivotal role of impaired bioenergetics is obvious in metabolic epilepsies, there is a gap of knowledge regarding secondary changes in metabolite patterns as a result of genetic Scn1a deficiency and ketogenic diet in the Dravet syndrome. METHODS: A comprehensive untargeted metabolomics analysis, along with assessment of epileptiform activity and behavioral tests, was completed in a Dravet mouse model. Data sets were compared between animals on a control and a ketogenic diet, and metabolic alterations associated with Dravet mice phenotype and ketogenic diet were identified. RESULTS: Hippocampal metabolomic data revealed complex alterations in energy metabolism with an effect of the genotype on concentrations of glucose and several glycolysis and tricarboxylic acid (TCA) cycle intermediates. Although low glucose, lactate, malate, and citrate concentrations became evident, the increase of several intermediates suggested a genotype-associated activation of catabolic processes with enhanced glycogenolysis and glycolysis. Moreover, we observed an impact on the glutamate/γ-aminobutyric acid (GABA)-glutamine cycle with reduced levels of all components along with a shift toward an increased GABA-to-glutamate ratio. Further alterations comprised a reduction in hippocampal levels of noradrenaline, corticosterone, and of two bile acids. SIGNIFICANCE: Considering that energy depletion can predominantly compromise the function of GABAergic interneurons, the changes in energy metabolism may contribute to seizure susceptibility and ictogenesis. They may also explain the therapeutic potential of the ketogenic diet, which aims to shift energy metabolism toward a more fat-based energy supply. Conversely, the increased GABA-to-glutamate ratio might serve as an endogenous compensatory mechanism, which can be further supported by GABAergic drugs, representing the mainstay of therapeutic management of Dravet syndrome. In view of a possible neuroprotective function of bile acids, it might be of interest to explore a possible therapeutic potential of bile acid-mediated therapies, already in discussion for neurodegenerative disorders.

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.

Nest-building performance in rats: impact of vendor, experience, and sex.

Nest building behavior has been intensely applied as a parameter for severity assessment in mice. In contrast, only a limited number of studies have reported nest building data from rats. Here, we assessed nest building in rats in two different facilities addressing the hypotheses that the vendor, previous experience with the nesting material as well as sex of the rats has an impact on the performance. Data from two study sites and three raters were compared to obtain information about the robustness of nest complexity scoring. The findings demonstrate a generally poor nest building performance in rats with a pronounced day-to-day fluctuation, and site-specific differences. Application of a newly developed scoring system resulted in an intermediate inter-rater reliability. Previous experience with the nesting material did not exert a consistent impact on nest complexity scores. Sex differences proved to depend on vendor and animal facility without consistent findings supporting a superior performance in female or male rats. In conclusion, our findings argue against a robust and consistent influence of sex and familiarity with the nesting material. The comparison between facilities suggests that local conditions need to be considered as influencing factors, which should be explored in more detail by future multicenter approaches. Considering the day-to-day fluctuation and the intermediate inter-rater reliability, we highly recommend to base nest complexity evaluation on means from several subsequent days analyzed by a group of experienced raters.

Defining body-weight reduction as a humane endpoint: a critical appraisal.

In many animal experiments scientists and local authorities define a body-weight reduction of 20% or more as severe suffering and thereby as a potential parameter for humane endpoint decisions. In this study, we evaluated distinct animal experiments in multiple research facilities, and assessed whether 20% body-weight reduction is a valid humane endpoint criterion in rodents. In most experiments (restraint stress, distinct models for epilepsy, pancreatic resection, liver resection, caloric restrictive feeding and a mouse model for Dravet syndrome) the animals lost less than 20% of their original body weight. In a glioma model, a fast deterioration in body weight of less than 20% was observed as a reliable predictor for clinical deterioration. In contrast, after induction of chronic diabetes or acute colitis some animals lost more than 20% of their body weight without exhibiting major signs of distress. In these two animal models an exclusive application of the 20% weight loss criterion for euthanasia might therefore result in an unnecessary loss of animals. However, we also confirmed that this criterion can be a valid parameter for defining the humane endpoint in other animal models, especially when it is combined with additional criteria for evaluating distress. In conclusion, our findings strongly suggest that experiment and model specific considerations are necessary for the rational integration of the parameter 'weight loss' in severity assessment schemes and humane endpoint criteria. A flexible implementation tailored to the experiment or intervention by scientists and authorities is therefore highly recommended.

Semi-automated generation of pictures for the Mouse Grimace Scale: A multi-laboratory analysis (Part 2).

The Mouse Grimace Scale (MGS) is an established method for estimating pain in mice during animal studies. Recently, an improved and standardized MGS set-up and an algorithm for automated and blinded output of images for MGS evaluation were introduced. The present study evaluated the application of this standardized set-up and the robustness of the associated algorithm at four facilities in different locations and as part of varied experimental projects. Experiments using the MGS performed at four facilities (F1-F4) were included in the study; 200 pictures per facility (100 pictures each rated as positive and negative by the algorithm) were evaluated by three raters for image quality and reliability of the algorithm. In three of the four facilities, sufficient image quality and consistency were demonstrated. Intraclass correlation coefficient, calculated to demonstrate the correlation among raters at the three facilities (F1-F3), showed excellent correlation. The specificity and sensitivity of the results obtained by different raters and the algorithm were analysed using Fisher's exact test (p < 0.05). The analysis indicated a sensitivity of 77% and a specificity of 64%. The results of our study showed that the algorithm demonstrated robust performance at facilities in different locations in accordance with the strict application of our MGS setup.

Toward evidence-based severity assessment in rat models with repeated seizures: II. Chemical post-status epilepticus model.

OBJECTIVE: Considering the complexity of neuronal circuits and their epilepsy-associated alterations, epilepsy models cannot be completely replaced by in vitro experimental approaches. Decisions about ethical approval of in vivo studies require a thorough weighing of the animal's burden and the benefit regarding the expected gain in knowledge. METHODS: Based on combined behavioral, biochemical, and physiological analyses, we assessed the impact on animal well-being and condition in different phases of the pilocarpine post-status epilepticus (SE) model in rats. RESULTS: As a consequence of SE, increased levels of impairment were evident in the early postinsult phase and late chronic phase, whereas only mild impairment was observed in the interim phase. Parameters that stood out as sensitive indicators of animal distress include burrowing, which proved to be affected throughout all experimental phases, saccharin preference, fecal corticosterone metabolites, heart rate, and heart rate variability. SIGNIFICANCE: The cumulative burden with temporary but not long-lasting phases of more pronounced impairment suggests a classification of severe as a basis for laboratory-specific prospective and retrospective evaluation. Among the parameters analyzed, burrowing behavior and saccharin preference stand out as candidate parameters that seem to be well suited to obtain information about animal distress in epileptogenesis models.

Toward evidence-based severity assessment in rat models with repeated seizures: III. Electrical post-status epilepticus model.

OBJECTIVE: Ethical approval of experiments in chronic epilepsy models requires a careful balancing of the expected gain-in-knowledge with the level of distress. Thus recommendations for evidence-based severity assessment and classification are urgently needed for preclinical epilepsy research. METHODS: Therefore, we have completed a comprehensive analysis of alterations in behavioral, biochemical, and physiological parameters in a rat electrical post-status epilepticus model. Selected parameters were repeatedly analyzed during different experimental phases to obtain information about the level of distress throughout the course of the model. RESULTS: Behavioral patterns comprised an increase in activity along with a reduction in risk assessment behavior, active social interaction, saccharin preference as well as nonessential, but evolutionary-determined behavior such as nest building and burrowing. Among the biochemical parameters, fecal corticosterone metabolites proved to be increased in different phases of the experiment. In the early post-insult phase, this increase was reflected by elevated serum corticosterone concentrations. Telemetric recordings demonstrated increases in home cage activity and heart rate in selected experimental phases but argued against relevant changes in heart rate variability. Comparison between animals with tethered or telemetric recordings including a principal component analysis revealed differences between both groups. SIGNIFICANCE: The present findings further confirm that burrowing behavior and saccharin preference might serve as valid parameters for severity assessment in chronic epilepsy models. Considering the course of alterations providing evidence for a more pronounced level of distress in the early phase following status epilepticus (SE), we suggest a classification of the electrical post-SE model as severe. This suggestion may serve as a guidance for laboratory-specific evaluations. Comparison between data from animals with tethered and telemetric recordings indicated an impact of the mode of recordings. However, further research is necessary to analyze the validity of telemetry as a putative refinement measure.

Impact of repeated kindled seizures on heart rate rhythms, heart rate variability, and locomotor activity in rats.

Although an impact of epilepsy on circadian rhythmicity is well-recognized, there are profound gaps in our understanding of the influence of seizures on diurnal rhythms. The effect on activity levels and heart rate is of particular interest as it might contribute to the disease burden. The kindling model with telemetric transmitter implants provides excellent opportunities to study the consequences of focal and generalized seizures under standardized conditions. Data from kindled rats with generalized seizures revealed an increase in activity and heart rate during the resting phase. Total and short-term heart rate variabilities were not affected by electrode implantation or seizure induction. Ictal alterations in heart rate associated with generalized seizures were characterized by a biphasic bradycardia with an immediate drop of heart rate followed by a transient normalization and a second more steady decrease. In conclusion, the findings demonstrate that once daily generalized seizures can exert significant effects on heart rate rhythms. Respective alterations in patients would be of relevance for patient counselling and therapeutic management. Occurrence of biphasic bradycardia associated with seizure induction suggests that the kindling model is suitable to study the consequences and the prevention of ictal bradycardia, which may pose patients at risk for sudden unexpected death.

Proteomic profiling of epileptogenesis in a rat model: Focus on cell stress, extracellular matrix and angiogenesis.

Information about epileptogenesis-associated changes in protein expression patterns is of particular interest for future selection of target and biomarker candidates. Bioinformatic analysis of proteomic data sets can increase our knowledge about molecular alterations characterizing the different phases of epilepsy development following an initial epileptogenic insult. Here, we report findings from a focused analysis of proteomic data obtained for the hippocampus and parahippocampal cortex samples collected during the early post-insult phase, latency phase, and chronic phase of a rat model of epileptogenesis. The study focused on proteins functionally associated with cell stress, cell death, extracellular matrix (ECM) remodeling, cell-ECM interaction, cell-cell interaction, angiogenesis, and blood-brain barrier function. The analysis revealed prominent pathway enrichment providing information about the complex expression alterations of the respective protein groups. In the hippocampus, the number of differentially expressed proteins declined over time during the course of epileptogenesis. In contrast, a peak in the regulation of proteins linked with cell stress and death as well as ECM and cell-cell interaction became evident at later phases during epileptogenesis in the parahippocampal cortex. The data sets provide valuable information about the time course of protein expression patterns during epileptogenesis for a series of proteins. Moreover, the findings provide comprehensive novel information about expression alterations of proteins that have not been discussed yet in the context of epileptogenesis. These for instance include different members of the lamin protein family as well as the fermitin family member 2 (FERMT2). Induction of FERMT2 and other selected proteins, CD18 (ITGB2), CD44 and Nucleolin were confirmed by immunohistochemistry. Taken together, focused bioinformatic analysis of the proteomic data sets completes our knowledge about molecular alterations linked with cell death and cellular plasticity during epileptogenesis. The analysis provided can guide future selection of target and biomarker candidates.