Cognitive deficits and brain myo-Inositol are early biomarkers of epileptogenesis in a rat model of epilepsy
Introduction
Epileptogenesis is a dynamic process of molecular, cellular and functional reorganization occurring in brain following precipitating events that lead to epilepsy (Pitkanen and Engel, 2014). Epilepsy is one of the most prevalent brain disorders affecting around 60 million people worldwide (Duncan et al., 2006). It is characterized by an enduring predisposition to generate spontaneous seizures often associated with cognitive and psychiatric comorbidities with negative social consequences. One major clinical need in epilepsy is to identify antiepileptogenesis treatments for preventing or arresting the development of the disease in patients who have been exposed to potentially epileptogenic brain insults, including status epilepticus (SE) (Holtkamp et al., 2005, Wagenman et al., 2014). The development of such treatments has been hampered by the lack of non-invasive biomarkers that could be used to identify the patients at-risk, thereby allowing to design affordable clinical studies (Engel et al., 2013, Pitkanen and Engel, 2014).
Our main goal was to test if cognitive deficits and astrocyte activation in seizure susceptible brain areas, both phenomena occurring in human epilepsy (Elger et al., 2004, Vezzani et al., 2012) and related animal models (Kleen et al., 2012, Vezzani et al., 2011), are biomarkers of epileptogenesis, thus predicting who is going to develop epilepsy after an acute brain injury. We used a well-established pilocarpine model of SE in 21 day-old rats where only a cohort of animals develops epilepsy (Marcon et al., 2009, Roch et al., 2002). This model mimics de novo SE in humans; SE is a relatively common clinical condition (10–41/100,000 population) (Betjemann and Lowenstein, 2015) with the majority of cases (54%) occurring in the absence of an antecedent diagnosis of epilepsy (Hesdorffer et al., 1998). 53% of patients (9/17) with de novo refractory SE were reported to develop epilepsy (Holtkamp et al., 2005) and in children with non-febrile convulsive SE subsequent epilepsy occurred in 13–74% of cases (Raspall-Chaure et al., 2006).
By focusing our analyses in the hippocampus, a key epileptogenic area in our animal model, we measured increased myo-Inositol (mIns) levels, a metabolite reflecting astrocyte activation (Brand et al., 1993) by in vivo proton magnetic resonance spectroscopy (1H-MRS) and cognitive deficits in the Morris Water Maze (MWM), a virtual version of which was recently developed in humans (Barkas et al., 2012). We found that both phenomena arise in rats before the onset of epilepsy, and predict which animals will develop the disease with high fidelity. Our results provide a proof-of-principle evidence of the potential predictive value of cognitive functions and mIns levels in seizure-prone brain areas for epilepsy development in individuals at high-risk following potential epileptogenic injuries.
Section snippets
Animals
Male Sprague–Dawley rats (Charles River, Calco, Italy) at postnatal day (PN) 21 (with PN1 defined as the day of birth) were used. The pups were housed with their dams at constant temperature (23 °C) and relative humidity (60%) with a fixed 12 h light–dark cycle and free access to food and water until weaning at PN21. Older animals were housed one per cage. For each experimental protocol described in Fig. 1, male pups were used from four independent litters. All experimental procedures were
Results
After grouping the animals at 7 months post-SE in epileptic and non-epileptic rats, based on the presence of spontaneous seizures and the ADT, we retrospectively analysed and compared their behavioral performance in the MWM (Fig. 2) and their respective mIns levels by 1H-MRS (Fig. 3) that were both assessed before the predicted time of epilepsy onset.
Discussion
We report the novel evidence that cognitive deficits in a spatial memory test together with astrocyte activation in the hippocampus, predict the development of epilepsy in a rat model of de novo SE with high accuracy. This model is highly valuable for determining factors associated with epileptogenesis since only a cohort of rats develop spontaneous seizures after an inciting event of similar severity and duration (Marcon et al., 2009, Roch et al., 2002). In accord, we found that only 70% of
Conclusions
This study provides the first proof-of-principle demonstration that assessment of cognitive abilities and 1H-MRS analysis of mIns in seizure-prone brain areas following potential epileptogenic injuries, may represent clinically meaningful biomarkers for early identification of individuals at high-risk for developing epilepsy (Betjemann and Lowenstein, 2015, Herman, 2002, Raspall-Chaure et al., 2006). Although our findings are related to a model of SE-induced epilepsy, increased mIns brain
Author contributions
R. Pascente injected rats with pilocarpine and conducted the behavioral and immunohistochemical experiments under the supervision of T. Ravizza; F. Frigerio implanted and monitored rats for seizure detection and measured ADT; M. Rizzi analysed EEG tracings; L. Porcu conducted statistical analysis of data; M. Boido performed stereological analysis; J. Davids and M. Zaben performed neurogenesis analysis with the supervision of W. P. Gray; D. Tolomeo and M. Filibian conducted 1H-MRS analysis and
Conflict of interest
The authors declare no competing financial interests.
Acknowledgements
This work was supported by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n. 602102 (EPITARGET; AV), Fondazione Monzino (5600) (AV), CURE (AV) and Epilepsy Research UK (F1204Zaben; WPG).
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Present address: Department of Physics, Università degli Studi di Pavia, Pavia, Italy.