RU486 mitigates Hippocampal Pathology Following Status Epilepticus

Status epilepticus induces rapid hyper-activation of the hypothalamo-pituitary-adrenocortical (HPA) axis. HPA axis hyperactivity results in excess exposure to high levels of circulating glucocorticoids, which are associated with neurotoxicity and depression-like behavior. These observations have led to the hypothesis that HPA axis dysfunction may exacerbate status epilepticus-induced brain injury. To test this hypothesis, we used the mouse pilocarpine model of epilepsy to determine whether use of the glucocorticoid receptor antagonist RU486 can attenuate hippocampal pathology following status epilepticus. Excess glucocorticoid secretion was evident one day after status epilepticus in the mice, preceding the development of spontaneous seizures (which can take weeks to develop). RU486 treatment blocked the SE-associated elevation of glucocorticoid levels in pilocarpine treated mice. RU486 treatment also mitigated the development of hippocampal pathologies induced by status epilepticus, reducing loss of hilar mossy cells and limiting pathological cell proliferation in the dentate hilus. Mossy cell loss and accumulation of ectopic hilar cells are positively correlated with epilepsy severity, suggesting that early treatment with glucocorticoid antagonists could have anti-epileptogenic effects

Functional disruption of stress modulatory circuits in a model of temporal lobe epilepsy.

Clinical data suggest that the neuroendocrine stress response is chronically dysregulated in a subset of patients with temporal lobe epilepsy (TLE), potentially contributing to both disease progression and the development of psychiatric comorbidities such as anxiety and depression. Whether neuroendocrine dysregulation and psychiatric comorbidities reflect direct effects of epilepsy-related pathologies, or secondary effects of disease burden particular to humans with epilepsy (i.e. social estrangement, employment changes) is not clear. Animal models provide an opportunity to dissociate these factors. Therefore, we queried whether epileptic mice would reproduce neuroendocrine and behavioral changes associated with human epilepsy. Male FVB mice were exposed to pilocarpine to induce status epilepticus (SE) and the subsequent development of spontaneous recurrent seizures. Morning baseline corticosterone levels were elevated in pilocarpine treated mice at 1, 7 and 10 weeks post-SE relative to controls. Similarly, epileptic mice had increased adrenal weight when compared to control mice. Exposure to acute restraint stress resulted in hypersecretion of corticosterone 30 min after the onset of the challenge. Anatomical analyses revealed reduced Fos expression in infralimbic and prelimbic prefrontal cortex, ventral subiculum and basal amygdala following restraint. No differences in Fos immunoreactivity were found in the paraventricular nucleus of the hypothalamus, hippocampal subfields or central amygdala. In order to assess emotional behavior, a second cohort of mice underwent a battery of behavioral tests, including sucrose preference, open field, elevated plus maze, 24h home-cage monitoring and forced swim. Epileptic mice showed increased anhedonic behavior, hyperactivity and anxiety-like behaviors. Together these data demonstrate that epileptic mice develop HPA axis hyperactivity and exhibit behavioral dysfunction. Endocrine and behavioral changes are associated with impaired recruitment of forebrain circuits regulating stress inhibition and emotional reactivity. Loss of forebrain control may underlie pronounced endocrine dysfunction and comorbid psychopathologies seen in temporal lobe epilepsy

Epileptic mice show anxiety-like and hyperactive behaviors.

<p>Epileptic mice show reduced percent time spent in the center of the A) open field and D) open arms of the elevated plus maze. The total distance travelled by epileptic mice in the B) open field and E) elevated plus maze is decreased relative to controls. Epileptic mice spent more time freezing (immobility >2 seconds) in the C) open field and in the F) elevated plus maze compared to their control counterparts. G) 24h activity monitoring demonstrates overall hyperactivity of epileptic mice relative to control mice. Shaded region indicates the time when all behavioral tests were performed. (*p < 0.05, **p < 0.01, ***p < 0.001). Data presented as mean ± SEM, n = 9 mice per group.</p

Reduced expression of Fos in stress regulatory regions in epileptic mice.

<p>Representative micrographs showing Fos (red) and NeuN (green) immunoreactivity in A) the prelimbic region of the medial prefrontal cortex (PFC), C) the basal amygdala and E) ventral subiculum. Top panels in each set show control mice while bottom panels are from epileptic mice. Epileptic mice show decreased Fos expression in the B) prelimbic and infralimbic region of the PFC, D) the basal subdivision of the amygdaloid nucleus and F) the ventral subiculum relative to control mice. *p < 0.05, **p<0.01. Data presented as mean ± SEM, n = 6–8 mice per group.</p

Experimental design.

<p>A) Experimental time line for cohorts 1 (top) and 2 (bottom). Baseline morning corticosterone (CORT) samples were taken at 1 and 7 weeks post-SE for Cohort 1 and at week 10 for Cohort 2. At week 8, mice from Cohort 1 were exposed to restraint and blood samples were taken at 0, 30, 60, and 120 minutes from the onset of the stressor. Cohort 2 mice began behavioral testing at week 6 (SPT, sucrose preference test; EPM, elevated plus maze; FST, forced-swim test). B) Brain regions analyzed for Fos expression are based on the Paxinos and Watson brain atlas. Abbreviations. IL, infralimbic prefrontal cortex; PL, prelimbic prefrontal cortex; hippocampal subdivisions include: CA1, CA3, DG, dentate gyrus; VS, ventral subiculum; amygdaloid complex: CeA, central amygdala; LA, lateral amygdala; BA, basal amygdala and PVN, paraventricular nucleus of the hypothalamus. Figure is modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197955#pone.0197955.ref026" target="_blank">26</a>].</p

Corticosterone hyper-secretion is present at baseline and stress-induced states in epileptic mice.

<p>A) Epileptic mice show increase levels of corticosterone (CORT) secretion following 30 minutes of restraint stress (gray region shows time in restrainer). B) Epileptic mice in cohort 1 show increased corticosterone baseline secretion at 1 and 7 weeks compared to control mice. C) Epileptic mice in cohort 2 show increased corticosterone baseline secretion at week 10 relative to controls. D) Adrenal weight of epileptic mice in cohort 2 is greater than control mice. (*p < 0.05, epileptic vs. control; ***p < 0.001). Data presented as mean ± SEM, Cohort 1 n = 6–8 mice per group, Cohort 2 n = 7–9 mice per group).</p

Epileptic mice show anhedonia and hyperactive behaviors.

<p>A) Sucrose preference was decreased in epileptic mice relative to controls. B) Epileptic mice showed increased active behaviors in the forced swim test. (*p < 0.05). Data presented as mean ± SEM, n = 9 mice per group.</p