Cognitive deficits and brain myo-Inositol are early biomarkers of epileptogenesis in a rat model of epilepsy

doi:10.1016/j.nbd.2016.05.001

Neurobiology of Disease

Volume 93, September 2016, Pages 146-155

https://doi.org/10.1016/j.nbd.2016.05.001 Get rights and content

Highlights

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Cognitive deficits and astrocyte activation arise before epilepsy onset
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Accelerated forgetting and reduced learning rate both predict the development of epilepsy
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Increased hippocampal myo-Inositol levels by in vivo imaging, an indicator of astrocyte activation, predicts epilepsy development
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These noninvasive measures are potentially relevant for early identification of individuals at high-risk for developing epilepsy

Abstract

One major unmet clinical need in epilepsy is the identification of therapies to prevent or arrest epilepsy development in patients exposed to a potential epileptogenic insult. 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. Our goal was to test the predictive value of cognitive deficits and brain astrocyte activation for the development of epilepsy following a potential epileptogenic injury. We used a model of epilepsy induced by pilocarpine-evoked status epilepticus (SE) in 21-day old rats where 60–70% of animals develop spontaneous seizures after around 70 days, although SE is similar in all rats. Learning was evaluated in the Morris water-maze at days 15 and 65 post-SE, each time followed by proton magnetic resonance spectroscopy for measuring hippocampal myo-Inositol levels, a marker of astrocyte activation. Rats were video-EEG monitored for two weeks at seven months post-SE to detect spontaneous seizures, then brain histology was done. Behavioral and imaging data were retrospectively analysed in epileptic rats and compared with non-epileptic and control animals. Rats displayed spatial learning deficits within three weeks from SE. However, only epilepsy-prone rats showed accelerated forgetting and reduced learning rate compared to both rats not developing epilepsy and controls. These deficits were associated with reduced hippocampal neurogenesis. myo-Inositol levels increased transiently in the hippocampus of SE-rats not developing epilepsy while this increase persisted until spontaneous seizures onset in epilepsy-prone rats, being associated with a local increase in S100β-positive astrocytes. Neuronal cell loss was similar in all SE-rats. Our data show that behavioral deficits, together with a non-invasive marker of astrocyte activation, predict which rats develop epilepsy after an acute injury. These measures have potential clinical relevance for identifying individuals at-risk for developing epilepsy following exposure to epileptogenic insults, and consequently, for designing adequately powered antiepileptogenesis trials.

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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CXCL1, a functional murine orthologue of the human chemokine CXCL8 (IL-8), and its CXCR1 and CXCR2 receptors were investigated in a murine model of acquired epilepsy developing following status epilepticus (SE) induced by intra-amygdala kainate. CXCL8 and its receptors were also studied in human temporal lobe epilepsy (TLE). The functional involvement of the chemokine in seizure generation and neuronal cell loss was assessed in mice using reparixin (formerly referred to as repertaxin), a non-competitive allosteric inhibitor of CXCR1/2 receptors.
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Pitkänen and Immonen (2014) reported that increased diffusion trace (T1σ) in the somatosensory and perirhinal cortices and hippocampus and increased T2 in the thalamus indicated rats with increased seizure susceptibility after pentylenetetrazol administration after lateral fluid-percussion induced TBI. More recently, Pascente et al. (2016) showed that increased hippocampal myo-inositol/total creatine ratio diagnosed P21 animals which were going to develop epilepsy after lithium-pilocarpine -induced SE from those that will not. Finally, human data from Friedman's laboratory showed that the area of the gadolinium leakage around the cortical lesion rather than the area of the cortical lesion itself indicated the presence of PTE (see Pitkänen et al., 2016a).
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¹: Present address: Department of Physics, Università degli Studi di Pavia, Pavia, Italy.

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Cognitive deficits and brain myo-Inositol are early biomarkers of epileptogenesis in a rat model of epilepsy

Highlights

Abstract

Introduction

Section snippets

Animals

Results

Discussion

Conclusions

Author contributions

Conflict of interest

Acknowledgements

J. Biol. Chem.

Lancet Neurol.

Trends Neurosci.

Exp. Neurol.

Lancet

Lancet Neurol.

Brain Res.

Brain Res.

Neurobiol. Dis.

Behav. Brain Res.

J. Biol. Chem.

Neurobiol. Dis.

Neurobiol. Dis.

Neurobiol. Dis.

Epilepsy Res.

Neurobiol Dis

Exp. Neurol.

Epilepsy Res.

Lancet Neurol.

J. Physiol. Paris

Seizure

Brain Res.

Proton spectroscopy detected myoinositol in children with traumatic brain injury

Pediatr. Res.

Fluoxetine restores spatial learning but not accelerated forgetting in mesial temporal lobe epilepsy

Brain

Multinuclear NMR studies on the energy metabolism of glial and neuronal cells

Dev. Neurosci.

Spatial memory, recognition memory, and the hippocampus

Proc. Natl. Acad. Sci. U. S. A.

Metabolic and cognitive response to human traumatic brain injury: a quantitative proton magnetic resonance study

J. Neurotrauma

Early deficits in spatial memory and theta rhythm in experimental temporal lobe epilepsy

J. Neurosci.

Long-term effects of status epilepticus in the immature brain are specific for age and model

Epilepsia

Low proliferation and differentiation capacities of adult hippocampal stem cells correlate with memory dysfunction in humans

Brain

Epidemiology of status epilepticus

J. Clin. Neurophysiol.

Epilepsy biomarkers

Epilepsia