Factors influencing the acute pentylenetetrazole‐induced seizure paradigm and a literature review

Abstract Objective To confirm the critical factors affecting seizure susceptibility in acute pentylenetetrazole (PTZ) mouse epilepsy models and evaluate the prior literature for these factors. Methods Serial cohorts of wild‐type mice administered intraperitoneal (IP)‐PTZ were aggregated and analyzed by multivariate logistic regression for the effect of sex, age, background strain, dose, and physiologic stress (i.e., EEG implantation and/or single‐housing) on seizure response. We assessed the reporting of these factors in a comprehensive literature review over the last 10 years (2010–2020). Results We conducted aggregated analysis of pooled data of 307 mice (220 C57BL/6J mice and 87 mixed background mice; 202 males, 105 females) with median age of 10 weeks (range: 6–49 weeks) with acute PTZ injection (dose range 40–65 mg/kg). Significance in multivariate analysis was found between seizures and increased PTZ dose (odds ratio (OR) 1.149, 95% confidence interval (CI) 1.102–1.205), older age (OR 1.1, 95% CI 1.041–1.170), physiologic stress (OR 17.36, 95% CI 7.349–44.48), and mixed background strain (OR 0.4725, 95% CI 0.2315–0.9345). Literature review identified 97 papers using acute PTZ‐seizure models. Age, housing, sex, and background were omitted by 61% (59/97), 51% (49/97), 18% (17/97), and 8% (8/97) papers, respectively. Only 17% of publications specified all four factors (16/97). Interpretation Our analysis and literature review demonstrate a critical gap in standardization of acute PTZ‐induced seizure paradigm in mice. We recommend that future studies specify and control for age, background strain, sex, and housing conditions of experimental animals.


Introduction
Rigor and reproducibility in preclinical epilepsy models is essential for understanding epilepsy and expediting antiseizure drug discovery. [1][2][3] Mouse models are particularly attractive for epilepsy research, as they can be genetically manipulated to recapitulate the genetic factors associated with many types of epilepsy. [4][5][6][7][8] In addition to genetic models, mice with single or spontaneous, recurrent seizures can be generated through the use of electrical stimulation or chemo-convulsants, such as pentylenetetrazole (PTZ), pilocarpine, and kainic acid. [9][10][11] PTZ specifically has been utilized to model epilepsy since the 1940s and has been a powerful tool for antiseizure drug discovery. 2,[12][13][14] While the mechanism of action is still not fully understood, PTZ is thought to act primarily as a GABA A receptor antagonist to induce seizures, and is employed in both acute and chronic models of epilepsy. 15,16 In acute paradigms, PTZ injection intraperitoneally (IP), subcutaneously (SubQ), or intravenously (IV) induces a stereotypic seizure response, following the Racine seizure scale and progressing through myoclonic jerking to generalized tonic-clonic seizures (GTCS). 17 There are multiple endpoints to evaluate seizure susceptibility in a PTZ paradigm, including seizure onset, latency, or duration; dosage threshold; Racine or other behavioral scales; electroencephalogram (EEG) characteristics; and mortality. Chronic epilepsy paradigms administer PTZ repeatedly to induce a kindling effect. 18,19 Acute PTZ model offers many benefits as a seizure paradigm, including its ease of administration, ability to evoke a full range of seizure types, and utility in drug screening. 20 However, PTZ has been used inconsistently to induce acute seizures, making it difficult to compare or replicate results across studies and evaluate potential treatments. There are many potential factors that could confound and lead to inconsistencies in prior studies. Strain differences in seizure susceptibility among inbred and outbred strains are well-documented. 21,22 Nonovariectomized females have a higher seizure threshold to PTZ-induced seizures, 23 but this difference may be dosedependent. 24 In rat models, sex and housing did not influence PTZ-induced seizures. 25 A recent study has demonstrated single-housing induces stress in mice and reduces seizure threshold to pilocarpine-induced seizures. 26 The effect of housing conditions on PTZ seizure threshold has not been tested in mice. In general, physiologic stress is known to increase seizure susceptibility, but the individual contributions are inconsistently documented in the literature. 27 EEG implantation increased seizures in the acute setting in a kainic acid seizure paradigm. 28 Furthermore, it is unclear if prior studies considered these factors in their preclinical research.
To better understand the influences of age, background strain, sex, and physiologic stress on acute PTZ-induced seizures, we evaluated aggregated seizure response data from several serial cohorts of IP-injected mice in the Experimental Neurophysiology Core at Boston Children's Hospital. We then systematically evaluated the literature from the past 10 years to assess if these critical factors were specified and controlled for in acute PTZ paradigm protocols across several studies. Our study and literature review together outline the important factors necessary to specify and control for in acute PTZ-induced seizure paradigms and serve as a framework for future studies using acute PTZ administration to model epilepsy in mice.

Mice
All mouse procedures were performed in accordance with the Guide for the Humane Use and Care of Laboratory Animals, and the studies included were approved by the Animal Care and Use Committee of Boston Children's Hospital. All mice were housed in a 12-h light-dark cycle, climate-and humidity-controlled room, with access to food and water ad libitum.
Littermates and sexes were divided evenly across experimental groups. Cohorts of wild-type control mice were included in this analysis, some of which have previously been published. 29,30 Wild-type mice on both pure C57BL/ 6J and mixed (129S4/SvJae, C57BL/6J, and CBA) backgrounds were used. 31 Ages of mice were grouped as follows, in accordance with widely accepted guidelines: pup (P0 to P21), juvenile (3-6 weeks), young adult (6-12 weeks), mature adult (13 weeks to 6 months), middleaged adult (10-14 months), and old-aged adult (over 18 months). 32 Mice were either socially housed (singlesex group-housing for entire life, 2-5 mice per cage) or singly housed (isolated for ≥1 week, as required in EEGmonitoring or behavioral experiments).
Acute PTZ seizure paradigm PTZ (Sigma-Aldrich, #P6500) was dissolved fresh before each experiment in 0.9% saline at 10 mg/mL. All mice were injected during the light phase of the light-dark cycle. Mice were weighed prior to dosing and injected IP (volume PTZ (mL) = weight (kg) 9 dose (mg/kg)/ 10 mg/mL). Post-injection, mice were placed in individual transparent cages and monitored for seizure over a 10-min period. After a 10-min observation period, mice were killed. Our primary endpoint was GTCS, as behaviorally indicated by loss of balance and continuous, uncontrollable jerking (Racine score of 5). A GTCS ended when the animal regained balance or righted itself and ceased jerking (post-ictal phase) or died. Start and finish time of GTCS and time of death were recorded.
We analyzed aggregated data from 22 both published and unpublished cohorts from the Experimental Neurophysiology Core at Boston Children's Hospital over a 7year period (2013-2020) using the same standardized method for PTZ-induced seizures. Because these cohorts were collected and measured in comparable ways and between-cohort heterogeneity is negligible, aggregated ª 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association analysis of the pooled data was performed. 33 The benefit of utilizing the larger pooled data set was to critically assess potential factors that influence seizure susceptibility with increased power. We included studies that injected single doses of IP PTZ following the previously outlined protocol, using GTCS as the primary endpoint. We evaluated the contributions of sex, age (young to middle-aged adults, range: 6-49 weeks), background strain (C57BL/6J v. mixed, as previously described), dose (40-65 mg/kg), and physiologic stress (housing: social v. single; EEG surgery: implanted v. not) to seizure response in an acute PTZ model.
Statistical analyses were performed using GraphPad Prism 8 and RStudio software. All factors in analysis were compared to seizure response. Univariate comparisons were performed by Pearson's chi-squared tests for categorical data (sex, stress, and background), Mann-Whitney U tests for continuous, non-parametric data (age and dose) and Spearman correlation. Fisher's exact test and chi-square test of trend were performed on subgroup analysis. Multivariate comparisons were performed by multivariate logistic regression analysis. A p-value of less than 0.05 was considered statistically significant.

Literature review
To identify studies that employed acute PTZ as a seizure paradigm, we performed a PubMed search for the 10 years (January 2010-May 2020) using the following search criteria: ( Papers were excluded post hoc if they were review articles, kindling models (repetitive injections of PTZ) otherwise not omitted, PTZ paradigms in zebrafish or rat models only, and/or evaluations of PTZ as a treatment only. Our search yielded 130 results, and 97 papers were included in our analysis.
Per reviewed manuscript, we recorded the age, background strain, sex, and housing of experimental mice, and route of PTZ administration, dose, and primary endpoint(s). We systematically evaluated if each of these critical factors were included in the study cohort. A factor was considered unspecified (uns.) if there was no mention of it or there was insufficient detail to replicate the study. Primary endpoints were grouped into categories for simplicity: (1) binomial seizure endpoint (GTCS, other seizure, death), (2) seizure feature (e.g., latency, duration), (3) behavioral scoring, (4) EEG characterization, (5) seizure threshold calculation, (6) other, or (7) undefined/unclear. Ages were grouped as previously explained.

Literature review of PTZ-induced seizures in mice
Our data highlight the importance of accounting for baseline characteristics in PTZ-induced seizure mouse models of epilepsy. We undertook a comprehensive literature review to evaluate prior studies using acute PTZ-induced seizures in mouse models. We identified 97 publications over the past 10 years (2010-2020). Our findings are summarized in Table 3 (full review in  25/27). In the studies utilizing the IP route, the most common primary endpoints were either binomial seizure/no seizure (27%, 26/97) or seizure scoring scales (26%, 25/97). Forty-nine percent (47/97) of papers included more than one primary seizure endpoint.

Discussion
The purpose of this study was two-fold: (1) to evaluate the influences of age, background strain, sex, and physiologic stress on seizure susceptibility in an acute PTZ epilepsy mouse model through aggregated analysis, and (2) to critically examine the way the field has accounted for these factors over the last 10 years. In our study, we identified dose, age, and stress as critical factors with significant influences on seizure susceptibility in an acute PTZ paradigm. In our literature review, we found that acute PTZ models are inconsistent across studies. Due to varying endpoints and most studies not explicitly stating confounding factors, it is difficult to compare or replicate results and evaluate potential treatments. Together, our aggregated analysis and comprehensive literature review demonstrate a critical gap in standardizing the acute PTZ-induced seizure paradigm to study epilepsy. It is likely our findings on age, background strain, sex, physiologic stress, and dose will generalize to other ages, strains, and PTZ routes of administration (IV and SubQ) not employed in our study, but more research is necessary to evaluate the role of factors we do not use in our labs.
The American Epilepsy Society (AES) and International League Against Epilepsy (ILAE) Translational Task Force have begun a systematic review of animal research in epilepsy. 34 This systematic review excludes acute seizure models. Therefore, we evaluated confounds of the acute PTZ-induced seizure method and evaluated the rigor of prior studies. Our initial goal was to perform a traditional meta-analysis of the prior literature to evaluate the confounding effects of critical factors (age, sex, physiologic stress, and background strain). However, in our initial literature review, we found the majority of papers omitting at least one of the four critical factors. We evaluated the importance of these factors in our cross-sectional analysis of the cohorts from our institution and confirmed the importance of these factors. The greatest strength of our study was an increased sample size and the standardization of the method used across our laboratories.
1 Pup: 0-3 weeks, Juvenile: 3-6 weeks, Young adult: 6-12 weeks, Mature adult: 3-6 months. 2 For example tonic seizures and myoclonic jerking. 3 That is latency, duration, or frequency. 4 For example Racine or other behavioral scoring. 5 That is a mathematical calculation of seizure threshold. models. Social isolation increased seizure susceptibility after pilocarpine-induced seizures. 26 In a rat study, housing influenced seizure susceptibility to bicuculline. 25 Future studies should explicitly evaluate the effect of social isolation in PTZ-induced seizures in multiple background strains of wild-type mice. In prior literature, housing conditions were not explicitly detailed in over half of the acute PTZ studies from the last decade. It is therefore crucial for the interpretation and replication of results that housing conditions are specified as either group (social) or single (isolated) when using the acute PTZ paradigm. A limitation of our study is that most of the animals were socially isolated before PTZ administration due to EEG electrode implantation surgery that involved acute exposure to anesthesia. The influence of EEG electrode implantation on seizure susceptibility in mice is poorly understood. Per lab protocol, implanted mice were postoperatively administered analgesics and allowed to heal for at least a week before any further experiments could be conducted 29,30,31 . One study employing a kainic acid seizure paradigm found that EEG implantation increased seizure endpoints (specifically mortality) regardless of experimental groups. 28 This paper has two key differences from our study, however: (1) they use juvenile mice, and (2) their postoperative recovery period is much shorter (3 days). They suggest that increased seizure susceptibility is due to inflammation, which may or may not resolve at a later time point. Our data point toward a trend to increased seizure susceptibility in mice that are socially isolated and/or implanted with EEG electrodes. While we cannot exclude the fact that other stressors may also contribute to increased seizure susceptibility, our evidence indicates a graded increase seizure susceptibility to social isolation and EEG implantation in a PTZ-seizure paradigm. Consistent with prior studies, we found stress increases seizure susceptibility. 27 Ultimately, more research is needed to understand the mechanisms by which EEG implantation and social isolation alter seizure susceptibility in mice.
In addition, we identified age as a significant factor in seizure susceptibility in both our univariate and multivariate analyses. Our findings on age are limited in scope, as 72% of our mice fall in the young adult category, and our age range is restricted to 6-49 weeks. Even with this restricted age range, we still found a significant influence of age on seizure susceptibility. At the same time, age was the most commonly omitted critical factor in the literature, with many papers omitting age all together and others only specifying the mice as "adult." Our results provide evidence that even small differences in age result in variability in seizure susceptibility. All studies should report the age range of mice used and restrict the range of ages as much as possible. The underlying mechanisms of seizure susceptibility across ages are not well-defined, and further research is needed in this area.
We did not identify sex as a significant contributor to seizure susceptibility in wild-type mice in an acute PTZ paradigm. Most studies did specify the sex of their experimental mice in their methods, and of these studies, a majority used only males. While we did not find sex differences in our study, it would be hasty to generalize these findings to all possible experimental designs, and, therefore, the sex breakdown of an experimental cohort is essential in any acute PTZ methods section. Sex differences are well-documented in aging and maturation of mice. 35 We did not track estrous cycles in this study, but previous studies suggest an influence of estrogen and estrous cycles on seizure susceptibility in rodent models. 36 Some papers in our literature review used this as justification for including only male mice in their studies. It is possible that any effects of estrous cycling were negated by our large sample size. It is also possible that there is no overt sex difference in untreated, wild-type mice with PTZ-induced seizures with a large sample size, but sex differences may be evident after treatments or genetic mutant models. Collectively, we are hesitant to conclude whether or not sex plays a role in PTZ-induced seizure susceptibility, as more nuanced effects may have been averaged out in our analysis.
Other studies have shown that background strain influences seizure presentation and susceptibility. 21,22 Our results are limited in their scope as we only evaluated C57BL/6J and a single mixed background strain. We found a significant difference in seizure susceptibility in the pure C57BL/6J and mixed background mice in our multivariate analysis. In our literature review, CD-1/Swiss Albino mice appear to have an increased seizure susceptibility with seizure rates of 100% in all studies reviewed. Additionally, 16 papers identified their mice as C57BL/6, which no longer indicates an actual strain. C57BL/6 requires a laboratory code at the end which indicates the substrain (e.g., J is The Jackson Laboratory and N is the NIH). 37,38 Based on our findings, we recommend that all studies employing PTZ as an acute paradigm specify fully the background strain of their experimental mice.

Conclusion
The cohorts included in our study used the most common acute PTZ method of administration (IP), primary endpoint (binomial seizure endpoint), and background strain (C57BL/6J), making our findings useful for a majority of the field. We show that dose, age, background strain, and physiologic stress all influence seizure susceptibility. Physiologic stressors of surgery and housing ª 2021 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association conditions influence PTZ-induced seizures, but the mechanisms of these effects remain to be determined. As proposed by the AES-ILAE report on common data elements, our data highlight the importance of including age, background strain, sex, and housing to improve the rigor and reproducibility of preclinical models of epilepsy. 39

Acknowledgments
This study was also supported by the BCH Neurodevelopmental Behavioral Core of the Intellectual and Developmental Disabilities Research Center (NIH U54 HD090255), the BCH Rosamund Stone Zander Translational Neuroscience Center, and the BCH Experimental Neurophysiology Core. C.Y. receives grant support from NIH 1K08NS107637, the Hearst Foundation, and Boston Children's Office of Faculty Development Career Development Fellowship. We thank all of the members of the Rotenberg and Sahin labs.

Conflicts of Interest
M.S. reports grant support from Novartis, Roche, Pfizer, Biogen, Ipsen, LAM Therapeutics, Astellas, Bridgebio and Quadrant Biosciences. He has served on Scientific Advisory Boards for Sage, Roche, Celgene, Aeovian, Regenxbio and Takeda. None of the other authors has any conflict of interest to disclose.

Ethical Publication Statement
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Supporting Information
Additional supporting information may be found online in the Supporting Information section at the end of the article. Table S1. Cohorts of mice used for multivariate analysis of variables related to PTZ-induced seizures. Table S2. Summary of literature review for intravenous and subcutaneous routes of PTZ-induced seizures in mice. Table S3. Full literature review separated by route of PTZ administration.