Sex and gonadectomy modify behavioral seizure susceptibility and mortality in a repeated low-dose kainic acid systemic injection paradigm in mice

Abstract

Objective: Sex differences in epilepsy appear driven in part due to effects of gonadal steroids, with varying results in experimental models based on species, strain, and method of seizure induction. Furthermore, removing a main source of these steroids via gonadectomy may impact seizure characteristics differently in males and females. Repeated low-dose kainic acid (RLDKA) systemic injection paradigms were recently shown to reliably induce status epilepticus (SE) and hippocampal histopathology in C57BL/6J mice. Here, we investigated whether seizure susceptibility in a RLDKA injection protocol exhibits a sex difference, and whether gonadectomy differentially influences response to this seizure induction paradigm in males and females.

Methods: Adult C57BL/6J mice were left gonad-intact as controls or gonadectomized (females: ovariectomized, OVX; males: orchidectomized, ORX). At least 2 weeks later, KA was injected i.p. every 30 minutes at 7.5 mg/kg or less until the animal reached SE, defined by at least 5 generalized seizures (GS, Racine stage 3 or higher). Parameters of susceptibility to GS induction, SE development, and mortality rates were quantified.

Results: No differences in seizure susceptibility or mortality were observed between control males and control females. ORX males exhibited increased susceptibility and reduced latency to both GS and SE, but OVX females exhibited increased susceptibility and reduced latency to SE only. However, ORX males, but not OVX females, exhibited strongly increased seizure-induced mortality.

Significance: The RLDKA protocol is notable for its efficacy in inducing SE and seizure-induced histopathology in C57BL/6J mice, the background for many transgenic strains in current use in epilepsy research. The present results indicate that this protocol may be beneficial for investigating the effects of gonadal hormone replacement on seizure susceptibility, mortality, and seizure-induced histopathology, and that gonadectomy unmasks sex differences in susceptibility to seizures and mortality not observed in gonad-intact controls.

Figure 1.. Body weight is increased at 2-4 weeks after gonadectomy in females but not males.

Individual values (circles) and mean ± SD (squares and lines, respectively) for body weight measured on the day of RLDKA injection. Red = control females (F); blue = control males (M); pink = OVX females; light blue = ORX males. *p<0.05, ***p<0.001

Figure 2.. Effect of gonadectomy on susceptibility to GS is most prominent in males.

A) Individual values (circles) and mean ± SD (squares and lines, respectively) for the number of i.p. kainic acid injections needed to reach GS (GS). Raw data are presented; for statistical analysis, the data were log-transformed to improve normality. B) Individual values and mean ± SD for the total amount of KA injected to initiate GS. C) Percentage of mice in each group with at least one GS by two (left) or three (right) KA injections. D) Individual values and mean ± SD for the latency in minutes to reach GS. Raw data are presented; for statistical analysis, the data were square root-transformed to improve normality. Red = control females (F); blue = control males (M); pink = OVX females; light blue = ORX males. *p<0.05, **p<0.01, ***p<0.001

Figure 3.. OVX females and ORX males exhibit increased susceptibility to status epilepticus (SE).

A) Individual values (circles) and mean ± SD (squares and lines, respectively) for the number of kainic acid injections needed to reach SE. B) Individual values and mean ± SD for the total amount of KA injected to initiate SE. Raw data are presented; for statistical analysis, the data were log-transformed to improve normality. C) Percentage of mice in each group that developed SE by two (left) or three (right) KA injections. D) Individual values and mean ± SD for the cumulative Racine scores of the first 5 GS (i.e., to SE criterion). Raw data are presented; for statistical analysis, the data were log-transformed to improve normality. E) Individual values and mean ± SD for the latency in minutes to reach SE. Raw data are presented; for statistical analysis, the data were square root-transformed to improve normality. Red = control females (F); blue = control males (M); pink = OVX females; light blue = ORX males. *p<0.05, **p<0.01, ***p<0.001

Figure 4.. ORX males exhibit increased seizure-related mortality.

Kaplan-Meier plot for percent survival over 300 min starting from the time of the first KA injection. **p<0.01, ***p<0.001