Prenatal transport stress, postnatal maternal behavior, and offspring sex differentially affect seizure susceptibility in young rats
Introduction
Pediatric epilepsy is a relatively common neurological disorder [1], and seizures are thought to affect approximately 2–5% of all children [2]. While there are a large number of recognized causal factors, a significant proportion of children with epilepsy still have an unknown etiology despite extensive investigations [3]. As such, many studies have focused on determining the factors underlying seizure susceptibility and epileptogenesis, necessitating the development of animal models.
Various strategies exist for development of these models including genetic manipulation, focal lesions, and brain injury (including various forms of insult such as chemical, electrical, or traumatic) to normal neural substrates, all in the pursuit of studying the process of acquired epilepsy [4]. Although highly informative, these models are not designed to examine either innate susceptibility or the process of spontaneous epileptogenesis, both of which imply that the process of neurodevelopment is compromised. In the present study, we examine the effects of altered neurodevelopment by manipulating critical perinatal factors (prenatal stress and postnatal maternal care) and assessing male and female offspring seizure susceptibility. Using such a neurodevelopmental approach, as opposed to models that utilize adult animals, provided much more power to make inferences about pediatric epilepsy; a phenomenon that has a well-described strong developmental stage specificity [5], [6], [7]. Further, a large percentage of studies restrict analysis to males, which is problematic for extrapolating findings to females as sex differences in many neurological disorders are now widely accepted [8], [9], [10].
Neural development proceeds according to a timed series of events [11]. Deviations from this programming result in aberrations of neurodevelopment ranging from structural abnormalities to cellular and subcellular functional impairment. Teratogenicity can result from a wide variety of factors that can be broadly divided into exogenous or endogenous etiologies. Endogenous teratogenicity can be further subdivided into factors related to maternal and fetal physiology and those related to maternal psychological factors. This is not a clean dichotomy as psychological stressors often involve a physiological response including activation of the hypothalamic–pituitary–adrenal axis [12]. While psychological stress undeniably affects the individual, studies have shown a significant impact of maternal psychological stress on fetal neurodevelopment in humans and animals (for reviews, see [13], [14]). Offspring effects are manifested as alterations in cognitive and behavioral function, as well as in structural and molecular changes in the brain [15], [16], [17], [18], [19]. The exact mechanisms underlying these effects are not fully known, but a role of the HPA axis and associated glucocorticoid release is supported by studies using administration of prenatal exogenous steroids noting similar changes in the neurobiology and behavior of the offspring [9]. However, as could be predicted, the changes associated with exogenous steroids are not exactly the same as those induced by exposure to stressors, a finding that may be a result of the latter also incorporating activation of the autonomic and immunological systems as part of the stress response.
Many different prenatal stress models have been used to examine developmental outcomes, all of which appear to have slightly different fetal and newborn outcomes. In another study in our lab, we examined the effect of prenatal immune challenge on newborn seizure susceptibility and found a significant difference in the seizure susceptibility of control postnatal day 14 (P14) pups as a function of the gestational timing of dam transport [20]. As this variable has significant potential to impact experimental outcomes, we sought to determine, more specifically, whether maternal-timed pregnant transport in and of itself modifies seizure susceptibility. We chose to focus on the effect of prenatal stress from a single day (gestational (G) day 9 or 16) as these two time points represent critical periods of brain development, hypothesizing that significant perturbations may induce different but long-lasting effects on neurodevelopment.
While prenatal stress is gaining a wider appreciation as a neurodevelopmental regulator, postnatal factors such as maternal behavior have also been shown to significantly impact neurodevelopment [6], [8], [21] and reshape prior experiences [22]. These effects have been noted at the cellular level as well as in terms of behavioral outcomes [22]. Although these effects have been largely investigated in animal models, the potential significance in human neurodevelopment is evident [15], [17]. However, the effect of the postnatal environment is also dependent somewhat on the prenatal experience not only in terms of the direct effect on the offspring but also with respect to the subsequent impact on maternal behavior [23]. Therefore, we monitored maternal behavior in all groups and analyzed its relationship to seizure susceptibility.
In summary, prior literature has clearly demonstrated that prenatal stress has the ability to significantly modify neurodevelopment. However, offspring outcomes have largely focused on behavioral, cognitive, or cellular changes, while less is known about the possible impact prenatal stress may have on seizure susceptibility and epilepsy. Therefore, we sought to investigate the sex-dependent effects of prenatal transport stress and postnatal maternal behavior on seizure susceptibility in P14 rat pups.
Section snippets
Animals and transport stress
Ten adult male and ten adult female Sprague–Dawley rats were ordered from Charles River (Quebec, Canada) prior to being bred in-house (IH). For IH bred dams, estrous cycle was determined daily (between 1030 h and 1100 h) with vaginal smears starting 2 days after arrival in the vivarium until 2 days before mating. The vaginal epithelial sample was collected and smeared on to SuperFrost slides, left at room temperature to dry, then fixed and stained using Diff-Quik staining kit (VWR, Canada). After
Control groups
There were no significant differences in litter size or pup weight as a function of maternal transport alone. However, other differences were noted in the pups at baseline and following stimuli (saline, LPS, KA, and FC) as determined by the amount of FosB-ir labeling (Figs. 1A and B). In the vDG (Fig. 1A), a significant difference was found between the transport groups [F(2,18) = 14.74, p < 0.01] where the IH baseline FosB-ir level was higher compared to G9 [t = 2.92, p < 0.05] and G16 [t = 5.36, p <
Discussion
In this study, we initially sought to determine whether maternal transport during pregnancy affected seizure susceptibility of offspring. Because maternal behavior plays an important role in neurodevelopment, we also examined the moderating effect of various maternal behaviors on the same outcome. Our results suggest that transporting dams during gestation alters pup characteristics, compared to those of IH-bred dams from the same strain and supplier and may, therefore, impact further
Conclusion
In essence, our results suggest that several factors including prenatal maternal stress, postnatal maternal behavior, offspring sex, and the seizure induction model affect seizure susceptibility in young rats. Although a routinely carried out event, transportation of pregnant dams introduces significant variability into experimental studies involving offspring development, and in our study, it influenced litter pup characteristics and baseline FosB-ir. Even more pronounced than the prenatal
Acknowledgments
The authors would like to thank Kay Murphy and Donna Goguen for their technical assistance, as well as Drs. Aylin Reid, James Heida, and Lisa Wright for conceptual help in various portions of the study. The study was funded by the William Dennis Chair and Research Fund in Pediatric Epilepsy (MJE), an IWK research grant (MJE), an Alberta Children's Hospital research grant (MJE, QJP), the Dalhousie Medical Research Foundation (MJE), and the Heart and Stroke Foundation of New Brunswick (RWC).
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