ABSTRACT
While neurosurgical interventions are frequently used in laboratory mice, refinement efforts to optimize analgesic management based on multimodal approaches appear to be rather limited. Therefore, we compared the efficacy and tolerability of combinations of the non-steroidal anti-inflammatory drug carprofen, a sustained-release formulation of the opioid buprenorphine, and the local anesthetic bupivacaine with carprofen monotherapy. Female and male C57BL/6J mice were subjected to isoflurane anesthesia and an intracranial electrode implant procedure. Given the multidimensional nature of postsurgical pain and distress, various physiological, behavioral, and biochemical parameters were applied for their assessment. The analysis revealed alterations in Neuro scores, home cage locomotion, body weight, nest building, mouse grimace scales, and fecal corticosterone metabolites. A composite measure scheme allowed the allocation of individual mice to severity classes. The comparison between groups failed to indicate the superiority of multimodal regimens over high-dose NSAID monotherapy. In conclusion, our findings confirmed the informative value of various parameters for assessment of pain and distress following neurosurgical procedures in mice. While all drug regimens were well tolerated in control mice, our data suggest that the total drug load should be carefully considered for perioperative management. Future studies would be of interest to assess potential synergies of drug combinations with lower doses of carprofen.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Mice, Inbred C57BL , Neurosurgical Procedures , Pain Management , Pain, Postoperative , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Mice , Male , Pain Management/methods , Female , Pain, Postoperative/drug therapy , Neurosurgical Procedures/adverse effects , Carbazoles/administration & dosage , Analgesia/methods , Bupivacaine/administration & dosage , Buprenorphine/administration & dosage , Analgesics, Opioid/administration & dosage , Drug Therapy, CombinationABSTRACT
OBJECTIVE: Given its key homeostatic role affecting mitochondria, ionotropic and metabotropic receptors, and voltage-gated ion channels, sigma-1 receptor (Sig1R) represents an interesting target for epilepsy management. Antiseizure effects of the positive allosteric modulator E1R have already been reported in acute seizure models. Although modulation of serotonergic neurotransmission is considered the main mechanism of action of fenfluramine, its interaction with Sig1R may be of additional relevance. METHODS: To further explore the potential of Sig1R as a target, we assessed the efficacy and tolerability of E1R and fenfluramine in two chronic mouse models, including an amygdala kindling paradigm and the intrahippocampal kainate model. The relative contribution of the interaction with Sig1R was analyzed using combination experiments with the Sig1R antagonist NE-100. RESULTS: Whereas E1R exerted pronounced dose-dependent antiseizure effects at well-tolerated doses in fully kindled mice, only limited effects were observed in response to fenfluramine, without a clear dose dependency. In the intrahippocampal kainate model, E1R failed to influence electrographic seizure activity. In contrast, fenfluramine significantly reduced the frequency of electrographic seizure events and their cumulative duration. Pretreatment with NE-100 reduced the effects of E1R and fenfluramine in the kindling model. Surprisingly, pre-exposure to NE-100 in the intrahippocampal kainate model rather enhanced and prolonged fenfluramine's antiseizure effects. SIGNIFICANCE: In conclusion, the kindling data further support Sig1R as an interesting target for novel antiseizure medications. However, it is necessary to further explore the preclinical profile of E1R in chronic epilepsy models with spontaneous seizures. Despite the rather limited effects in the kindling paradigm, the findings from the intrahippocampal kainate model suggest that it is of interest to further assess a possible broad-spectrum potential of fenfluramine.
Subject(s)
Disease Models, Animal , Epilepsy , Fenfluramine , Kindling, Neurologic , Receptors, sigma , Sigma-1 Receptor , Animals , Receptors, sigma/antagonists & inhibitors , Receptors, sigma/drug effects , Mice , Kindling, Neurologic/drug effects , Fenfluramine/pharmacology , Epilepsy/drug therapy , Male , Anticonvulsants/pharmacology , Anticonvulsants/therapeutic use , Dose-Response Relationship, Drug , Piperazines/pharmacology , Piperazines/therapeutic use , Amygdala/drug effects , Amygdala/physiopathology , Hippocampus/drug effects , Chronic Disease , Kainic Acid/pharmacology , Mice, Inbred C57BLABSTRACT
Objective: Heterozygous mutations within the voltage-gated sodium channel α subunit (SCN1A) are responsible for the majority of cases of Dravet syndrome (DS), a severe developmental and epileptic encephalopathy. Development of novel therapeutic approaches is mandatory in order to directly target the molecular consequences of the genetic defect. The aim of the present study was to investigate whether cis-acting long non-coding RNAs (lncRNAs) of SCN1A are expressed in brain specimens of children and adolescent with epilepsy as these molecules comprise possible targets for precision-based therapy approaches. Methods: We investigated SCN1A mRNA expression and expression of two SCN1A related antisense RNAs in brain tissues in different age groups of pediatric non-Dravet patients who underwent surgery for drug resistant epilepsy. The effect of different antisense oligonucleotides (ASOs) directed against SCN1A specific antisense RNAs on SCN1A expression was tested. Results: The SCN1A related antisense RNAs SCN1A-dsAS (downstream antisense, RefSeq identifier: NR_110598) and SCN1A-usAS (upstream AS, SCN1A-AS, RefSeq identifier: NR_110260) were widely expressed in the brain of pediatric patients. Expression patterns revealed a negative correlation of SCN1A-dsAS and a positive correlation of lncRNA SCN1A-usAS with SCN1A mRNA expression. Transfection of SK-N-AS cells with an ASO targeted against SCN1A-dsAS was associated with a significant enhancement of SCN1A mRNA expression and reduction in SCN1A-dsAS transcripts. Conclusion: These findings support the role of SCN1A-dsAS in the suppression of SCN1A mRNA generation. Considering the haploinsufficiency in genetic SCN1A related DS, SCN1A-dsAS is an interesting target candidate for the development of ASOs (AntagoNATs) based precision medicine therapeutic approaches aiming to enhance SCN1A expression in DS.
ABSTRACT
In animal-based research, welfare assessments are essential for ethical and legal reasons. However, accurate assessment of suffering in laboratory animals is often complicated by the multidimensional character of distress and pain and the associated affective states. The present study aimed to design and validate multidimensional composite measure schemes comprising behavioral and biochemical parameters based on a bioinformatics approach. Published data sets from induced and genetic mouse models of neurological and psychiatric disorders were subjected to a bioinformatics workflow for cross-model analyses. ROC analyses pointed to a model-specific discriminatory power of selected behavioral parameters. Principal component analyses confirmed that the composite measure schemes developed for adult or young mice provided relevant information with the level of group separation reflecting the expected severity levels. Finally, the validity of the composite measure schemes developed for adult and young mice was further confirmed by k-means-based clustering as a basis for severity classification. The classification systems allowed the allocation of individual animals to different severity levels and a direct comparison of animal groups and other models. In conclusion, the bioinformatics approach confirmed the suitability of the composite measure schemes for evidence-based comparative severity assessment in adult and young mice. In particular, we demonstrated that the composite measure schemes provide a basis for an individualized severity classification in control and experimental groups allowing direct comparison of severity levels across different induced or genetic models. An online tool (R package) is provided, allowing the application of the bioinformatics approach to severity assessment data sets regardless of the parameters or models used. This tool can also be used to validate refinement measures.
Subject(s)
Mental Disorders , Humans , Adult , Mice , Animals , Animals, Laboratory , EmotionsABSTRACT
The forced swim test (FST) is a traditional assay, which has been used for more than 40 years to assess antidepressant effects of novel drug candidates. In recent years, a debate about the test has focused on the assumption that the FST is highly aversive and burdening for the animals because of the earlier anthropomorphic interpretation and designation as a "behavioral despair test". The Directive 2010/63/EU and the German Animal Welfare law require a prospective severity classification of the planned experimental procedures. Still, an objective examination of the animals' burden in this test has not been performed yet. To fill this gap, we conducted an evidence-based severity assessment of the forced swim test in rats according to a 'standard protocol' with a water temperature of 25°C. We examined parameters representing the physiological and the affective state, and natural as well as locomotion-associated behaviors in three separate experiments to reflect as many dimensions as possible of the animal's condition in the test. Hypothermia was the only effect observed in all animals exposed to the FST when using this standard protocol. Additional adverse effects on body weight, food consumption, and fecal corticosterone metabolite concentrations occurred in response to administration of the antidepressant imipramine, which is frequently used as positive control when testing for antidepressant effects of new substances. We conclude that this version of the FST itself is less severe for the animals than assumed, and we suggest a severity classification of 'moderate' because of the acute and short-lasting effects of hypothermia. To refine the FST according to the 3Rs, we encourage confirming the predictive validity in warmer water temperatures to allow the rats to maintain physiological body temperature.
Subject(s)
Hypothermia , Rats , Animals , Prospective Studies , Antidepressive Agents/pharmacology , Imipramine/pharmacology , Swimming , Water/pharmacology , Behavior, Animal/physiologyABSTRACT
Many potentially noxious interventions are performed on chicken embryos in research and in the poultry industry. It is therefore essential and in the interest of animal welfare to be able to precisely define the point at which a chicken embryo is capable of nociception in ovo. The present part III of a comprehensive study examined the movements of developing chicken embryos with the aim of identifying behavioral responses to a noxious stimulus. For this purpose, a noxious mechanical stimulus and a control stimulus were applied in a randomized order. The recorded movements of the embryos were evaluated using the markerless pose estimation software DeepLabCut and manual observations. After the application of the mechanical stimulus, a significant increase in beak movement was identified in 15- to 18-day-old embryos. In younger embryos, no behavioral changes related to the noxious stimulus were observed. The presented results indicate that noxious mechanical stimuli at the beak base evoke a nocifensive reaction in chicken embryos starting at embryonic day 15.
ABSTRACT
Although it is assumed that chicken embryos acquire the capacity for nociception while developing in the egg, an exact time point has not yet been specified. The present research was an exploratory study aiming to determine when the capacity of nociception emerges during embryonic development in chickens. Changes in blood pressure and heart rate (HR) in response to a noxious mechanical stimulus at the base of the beak versus a light touch on the beak were examined in chicken embryos between embryonic days (EDs) 7 and 18. Mean arterial pressure (MAP) was the most sensitive parameter for assessing cardiovascular responses. Significant changes in MAP in response to a noxious stimulus were detected in embryos at ED16 to ED18, whereas significant changes in HR were observed at ED17 and ED18. Infiltration anesthesia with the local anesthetic lidocaine significantly reduced the response of MAP on ED18, so the measured cardiovascular changes may be interpreted as nociceptive responses.
ABSTRACT
Despite progress in the development of anti-seizure medications (ASMs), one third of people with epilepsy have drug-resistant epilepsy (DRE). The working definition of DRE, proposed by the International League Against Epilepsy (ILAE) in 2010, helped identify individuals who might benefit from presurgical evaluation early on. As the incidence of DRE remains high, the TASK1 workgroup on DRE of the ILAE/American Epilepsy Society (AES) Joint Translational Task Force discussed the heterogeneity and complexity of its presentation and mechanisms, the confounders in drawing mechanistic insights when testing treatment responses, and barriers in modeling DRE across the lifespan and translating across species. We propose that it is necessary to revisit the current definition of DRE, in order to transform the preclinical and clinical research of mechanisms and biomarkers, to identify novel, effective, precise, pharmacologic treatments, allowing for earlier recognition of drug resistance and individualized therapies.
Subject(s)
Drug Resistant Epilepsy , Epilepsy , Humans , United States , Epilepsy/drug therapy , Drug Resistant Epilepsy/drug therapy , Drug Resistance , Advisory Committees , IncidenceABSTRACT
This study presents a novel concept for a smart home cage design, tools, and software used to monitor the physiological parameters of mice and rats in animal-based experiments. The proposed system focuses on monitoring key clinical parameters, including heart rate, respiratory rate, and body temperature, and can also assess activity and circadian rhythm. As the basis of the smart home cage system, an in-depth analysis of the requirements was performed, including camera positioning, imaging system types, resolution, frame rates, external illumination, video acquisition, data storage, and synchronization. Two different camera perspectives were considered, and specific camera models, including two near-infrared and two thermal cameras, were selected to meet the requirements. The developed specifications, hardware models, and software are freely available via GitHub. During the first testing phase, the system demonstrated the potential of extracting vital parameters such as respiratory and heart rate. This technology has the potential to reduce the need for implantable sensors while providing reliable and accurate physiological data, leading to refinement and improvement in laboratory animal care.
Subject(s)
Animal Experimentation , Rodentia , Rats , Animals , Animal Husbandry , Body Temperature , TelemetryABSTRACT
The lack of therapeutics that prevent the development of epilepsy, improve disease prognosis or overcome drug resistance represents an unmet clinical need in veterinary as well as in human medicine. Over the past decade, experimental studies and studies in human epilepsy patients have demonstrated that neuroinflammatory processes are involved in epilepsy development and play a key role in neuronal hyperexcitability that underlies seizure generation. Targeting neuroinflammatory signaling pathways may provide a basis for clinically relevant disease-modification strategies in general, and moreover, could open up new therapeutic avenues for human and veterinary patients with drug-resistant epilepsy. A sound understanding of the neuroinflammatory mechanisms underlying seizure pathogenesis in canine patients is therefore essential for mechanism-based discovery of selective epilepsy therapies that may enable the development of new disease-modifying treatments. In particular, subgroups of canine patients in urgent needs, e.g. dogs with drug-resistant epilepsy, might benefit from more intensive research in this area. Moreover, canine epilepsy shares remarkable similarities in etiology, disease manifestation, and disease progression with human epilepsy. Thus, canine epilepsy is discussed as a translational model for the human disease and epileptic dogs could provide a complementary species for the evaluation of antiepileptic and antiseizure drugs. This review reports key preclinical and clinical findings from experimental research and human medicine supporting the role of neuroinflammation in the pathogenesis of epilepsy. Moreover, the article provides an overview of the current state of knowledge regarding neuroinflammatory processes in canine epilepsy emphasizing the urgent need for further research in this specific field. It also highlights possible functional impact, translational potential and future perspectives of targeting specific inflammatory pathways as disease-modifying and multi-target treatment options for canine epilepsy.
Subject(s)
Dog Diseases , Drug Resistant Epilepsy , Epilepsy , Dogs , Humans , Animals , Neuroinflammatory Diseases/veterinary , Epilepsy/veterinary , Epilepsy/drug therapy , Anticonvulsants/therapeutic use , Seizures/veterinary , Drug Resistant Epilepsy/veterinary , Dog Diseases/drug therapy , Dog Diseases/etiologyABSTRACT
Animal research has always been crucial for various medical and scientific breakthroughs, providing information on disease mechanisms, genetic predisposition to diseases, and pharmacological treatment. However, the use of animals in medical research is a source of great controversy and ongoing debate in modern science. To ensure a high level of bioethics, new guidelines have been adopted by the EU, implementing the 3R principles to replace animal testing wherever possible, reduce the number of animals per experiment, and refine procedures to minimize stress and pain. Supporting these guidelines, this article proposes an improved approach for unobtrusive, continuous, and automated monitoring of the respiratory rate of laboratory rats. It uses the cyclical expansion and contraction of the rats' thorax/abdominal region to determine this physiological parameter. In contrast to previous work, the focus is on unconstrained animals, which requires the algorithms to be especially robust to motion artifacts. To test the feasibility of the proposed approach, video material of multiple rats was recorded and evaluated. High agreement was obtained between RGB imaging and the reference method (respiratory rate derived from electrocardiography), which was reflected in a relative error of 5.46%. The current work shows that camera-based technologies are promising and relevant alternatives for monitoring the respiratory rate of unconstrained rats, contributing to the development of new alternatives for a continuous and objective assessment of animal welfare, and hereby guiding the way to modern and bioethical research.
ABSTRACT
Experimental craniotomies are a common surgical procedure in neuroscience. Because inadequate analgesia appears to be a problem in animal-based research, we conducted this review and collected information on management of craniotomy-associated pain in laboratory mice and rats. A comprehensive search and screening resulted in the identification of 2235 studies, published in 2009 and 2019, describing craniotomy in mice and/or rats. While key features were extracted from all studies, detailed information was extracted from a random subset of 100 studies/year. Reporting of perioperative analgesia increased from 2009 to 2019. However, the majority of studies from both years did not report pharmacologic pain management. Moreover, reporting of multimodal treatments remained at a low level, and monotherapeutic approaches were more common. Among drug groups, reporting of pre- and postoperative administration of non-steroidal anti-inflammatory drugs, opioids, and local anesthetics in 2019 exceeded that of 2009. In summary, these results suggest that inadequate analgesia and oligoanalgesia are persistent issues associated with experimental intracranial surgery. This underscores the need for intensified training of those working with laboratory rodents subjected to craniotomies. Systematic review registration: https://osf.io/7d4qe.
ABSTRACT
Drug resistance continues to be a major clinical problem in the therapeutic management of canine epilepsies with substantial implications for quality of life and survival times. Experimental and clinical data from human medicine provided evidence for relevant contributions of intrinsic severity of the disease as well as alterations in pharmacokinetics and -dynamics to failure to respond to antiseizure medications. In addition, several modulatory factors have been identified that can be associated with the level of therapeutic responses. Among others, the list of potential modulatory factors comprises genetic and epigenetic factors, inflammatory mediators, and metabolites. Regarding data from dogs, there are obvious gaps in knowledge when it comes to our understanding of the clinical patterns and the mechanisms of drug-resistant canine epilepsy. So far, seizure density and the occurrence of cluster seizures have been linked with a poor response to antiseizure medications. Moreover, evidence exists that the genetic background and alterations in epigenetic mechanisms might influence the efficacy of antiseizure medications in dogs with epilepsy. Further molecular, cellular, and network alterations that may affect intrinsic severity, pharmacokinetics, and -dynamics have been reported. However, the association with drug responsiveness has not yet been studied in detail. In summary, there is an urgent need to strengthen clinical and experimental research efforts exploring the mechanisms of resistance as well as their association with different etiologies, epilepsy types, and clinical courses.
Subject(s)
Dog Diseases , Drug Resistant Epilepsy , Epilepsy , Dogs , Animals , Humans , Anticonvulsants/therapeutic use , Quality of Life , Epilepsy/drug therapy , Epilepsy/veterinary , Seizures/veterinary , Drug Resistant Epilepsy/veterinary , Dog Diseases/drug therapyABSTRACT
Growing concerns over rigor and reproducibility of preclinical studies, including consistency across laboratories and translation to clinical populations, have triggered efforts to harmonize methodologies. This includes the first set of preclinical common data elements (CDEs) for epilepsy research studies, as well as Case Report Forms (CRFs) for widespread use in epilepsy research. The General Pharmacology Working Group of the ILAE/AES Task Force (TASK3-WG1A) has continued in this effort by adapting and refining CDEs/CRFs to address specific study design areas as they relate to preclinical drug screening: general pharmacology, pharmacokinetics (PK) and pharmacodynamics (PD), and tolerability. This work has expanded general pharmacology studies to include dose records, PK/PD, tolerability, and elements of rigor and reproducibility. Tolerability testing CRFs included rotarod and Irwin/Functional Observation Battery (FOB) assays. The material provided in the form of CRFs can be delivered for widespread use within the epilepsy research community.
ABSTRACT
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.
Subject(s)
Epilepsy , MicroRNAs , Status Epilepticus , Rats , Animals , MicroRNAs/genetics , MicroRNAs/metabolism , Rats, Sprague-Dawley , Epilepsy/genetics , Epilepsy/metabolism , Status Epilepticus/genetics , Status Epilepticus/metabolism , Biomarkers , Hippocampus/metabolismABSTRACT
Epilepsy is a challenging multifactorial disorder with a complex genetic background. Our current understanding of the pathophysiology and treatment of epilepsy has substantially increased due to animal model studies, including canine studies, but additional basic and clinical research is required. Drug-resistant epilepsy is an important problem in both dogs and humans, since seizure freedom is not achieved with the available antiseizure medications. The evaluation and exploration of pharmacological and particularly non-pharmacological therapeutic options need to remain a priority in epilepsy research. Combined efforts and sharing knowledge and expertise between human medical and veterinary neurologists are important for improving the treatment outcomes or even curing epilepsy in dogs. Such interactions could offer an exciting approach to translate the knowledge gained from people and rodents to dogs and vice versa. In this article, a panel of experts discusses the similarities and knowledge gaps in human and animal epileptology, with the aim of establishing a common framework and the basis for future translational epilepsy research.
Subject(s)
Dog Diseases , Drug Resistant Epilepsy , Epilepsy , Neurology , Humans , Animals , Dogs , Dog Diseases/drug therapy , Epilepsy/veterinary , Drug Resistant Epilepsy/veterinary , Treatment Outcome , Anticonvulsants/therapeutic useABSTRACT
Dravet syndrome is a severe developmental and epileptic encephalopathy mostly caused by heterozygous mutation of the SCN1A gene encoding the voltage-gated sodium channel α subunit Nav1.1. Multiple seizure types, cognitive deterioration, behavioral disturbances, ataxia, and sudden unexpected death associated with epilepsy are a hallmark of the disease. Recently approved antiseizure medications such as fenfluramine and cannabidiol have been shown to reduce seizure burden. However, patients with Dravet syndrome are still medically refractory in the majority of cases, and there is a high demand for new therapies aiming to improve behavioral and cognitive outcome. Drug-repurposing approaches for SCN1A-related Dravet syndrome are currently under investigation (i.e., lorcaserin, clemizole, and ataluren). New therapeutic concepts also arise from the field of precision medicine by upregulating functional SCN1A or by activating Nav1.1. These include antisense nucleotides directed against the nonproductive transcript of SCN1A with the poison exon 20N and against an inhibitory noncoding antisense RNA of SCN1A. Gene therapy approaches such as adeno-associated virus-based upregulation of SCN1A using a transcriptional activator (ETX101) or CRISPR/dCas technologies show promising results in preclinical studies. Although these new treatment concepts still need further clinical research, they offer great potential for precise and disease modifying treatment of Dravet syndrome.
Subject(s)
Epilepsies, Myoclonic , Epilepsy , Neurodevelopmental Disorders , Humans , NAV1.1 Voltage-Gated Sodium Channel/genetics , Epilepsies, Myoclonic/drug therapy , Epilepsies, Myoclonic/genetics , Epilepsy/genetics , SeizuresABSTRACT
INTRODUCTION: Chronic epilepsy models require neurosurgical procedures including depth electrode implants. The intrahippocampal kainate model is a frequently used chronic paradigm, which is based on chemoconvulsant administration and status epilepticus induction during the surgical procedure. This experimental approach raises the question of the extent to which this approach affects postsurgical recovery. In addition to the short- and long-term impact of the surgical intervention, a potential impact of highly frequent electrographic seizure events needs to be considered in the context of severity assessment. METHODS: Various behavioral, biochemical, and telemetric parameters were analyzed in four experimental groups of mice: 1st naive, 2nd with transmitter implants, 3rd with transmitter and electrode implants, and 4th with transmitter implants, electrode implants, and kainate-induced status epilepticus. RESULTS: During the early postsurgical phase, transmitter implants caused a transient impact on Mouse Grimace scores and intragroup increase of fecal corticosterone metabolites. Additional craniotomy was associated with an influence on total heart rate variability and fecal corticosterone metabolites. Heart rate and Irwin score increases as well as a prolonged increase in Mouse Grimace scores pointed to an added burden related to the induction of a nonconvulsive status epilepticus. Data from the chronic phase argued against a relevant influence of frequent electrographic seizures on behavioral patterns, fecal corticosterone metabolites, heart rate, and its variability. However, Irwin scores indicated long-term changes in some animals with increased reactivity, body tone, and Straub tail. Interestingly, selected behavioral and telemetric data from the early post-status epilepticus phase correlated with the frequency of electrographic seizure events in the chronic phase. CONCLUSION: In conclusion, our findings argue against the pronounced impact of highly frequent electrographic seizures on the well-being of mice. However, an increased level of nervousness in a subgroup of animals should be considered for handling procedures and refinement measures. In the early postsurgical phase, several parameters indicate an influence of the interventions with evidence that the nonconvulsive status epilepticus can negatively affect the recovery. Thus, the development and validation of refinement efforts should focus on this experimental phase. Finally, the datasets suggest that simple readout parameters may predict the long-term consequences of the epileptogenic insult. Respective biomarker candidates require further validation in the follow-up studies in models with subgroups of animals with or without epilepsy development.
Subject(s)
Epilepsy , Status Epilepticus , Mice , Animals , Kainic Acid/adverse effects , Corticosterone , Seizures/chemically induced , Status Epilepticus/chemically induced , Disease Models, AnimalABSTRACT
The influence of protocol standardization between laboratories on their replicability of preclinical results has not been addressed in a systematic way. While standardization is considered good research practice as a means to control for undesired external noise (i.e., highly variable results), some reports suggest that standardized protocols may lead to idiosyncratic results, thus undermining replicability. Through the EQIPD consortium, a multi-lab collaboration between academic and industry partners, we aimed to elucidate parameters that impact the replicability of preclinical animal studies. To this end, 3 experimental protocols were implemented across 7 laboratories. The replicability of results was determined using the distance travelled in an open field after administration of pharmacological compounds known to modulate locomotor activity (MK-801, diazepam, and clozapine) in C57BL/6 mice as a worked example. The goal was to determine whether harmonization of study protocols across laboratories improves the replicability of the results and whether replicability can be further improved by systematic variation (heterogenization) of 2 environmental factors (time of testing and light intensity during testing) within laboratories. Protocols were tested in 3 consecutive stages and differed in the extent of harmonization across laboratories and standardization within laboratories: stage 1, minimally aligned across sites (local protocol); stage 2, fully aligned across sites (harmonized protocol) with and without systematic variation (standardized and heterogenized cohort); and stage 3, fully aligned across sites (standardized protocol) with a different compound. All protocols resulted in consistent treatment effects across laboratories, which were also replicated within laboratories across the different stages. Harmonization of protocols across laboratories reduced between-lab variability substantially compared to each lab using their local protocol. In contrast, the environmental factors chosen to introduce systematic variation within laboratories did not affect the behavioral outcome. Therefore, heterogenization did not reduce between-lab variability further compared to the harmonization of the standardized protocol. Altogether, these findings demonstrate that subtle variations between lab-specific study protocols may introduce variation across independent replicate studies even after protocol harmonization and that systematic heterogenization of environmental factors may not be sufficient to account for such between-lab variation. Differences in replicability of results within and between laboratories highlight the ubiquity of study-specific variation due to between-lab variability, the importance of transparent and fine-grained reporting of methodologies and research protocols, and the importance of independent study replication.