Elsevier

Brain Research

Volume 1652, 1 December 2016, Pages 1-13
Brain Research

2Research report
Levetiracetam treatment influences blood-brain barrier failure associated with angiogenesis and inflammatory responses in the acute phase of epileptogenesis in post-status epilepticus mice

https://doi.org/10.1016/j.brainres.2016.09.038Get rights and content

Highlights

  • Increased BBB permeability was caused by the neovascularization in the acute phase after SE.

  • Pro-inflammatory responses in hippocampus enhanced in the acute phase after SE.

  • The angiogenesis and pro-inflammatory responses coincided with the BBB leakage in hippocampus.

  • LEV inhibited the BBB failure associated with angiogenesis and pro-inflammatory responses.

  • LEV may be key to developing new prophylactic therapies for post-brain insult epilepsy.

Abstract

Our previous study showed that treatment with levetiracetam (LEV) after status epilepticus (SE) termination by diazepam might prevent the development of spontaneous recurrent seizures via the inhibition of neurotoxicity induced by brain edema events. In the present study, we determined the possible molecular and cellular mechanisms of LEV treatment after termination of SE. To assess the effect of LEV against the brain alterations after SE, we focused on blood-brain barrier (BBB) dysfunction associated with angiogenesis and brain inflammation. The consecutive treatment of LEV inhibited the temporarily increased BBB leakage in the hippocampus two days after SE. At the same time point, the LEV treatment significantly inhibited the increase in the number of CD31-positive endothelial immature cells and in the expression of angiogenic factors. These findings suggested that the increase in neovascularization led to an increase in BBB permeability by SE-induced BBB failure, and these brain alterations were prevented by LEV treatment. Furthermore, in the acute phase of the latent period, pro-inflammatory responses for epileptogenic targets in microglia and astrocytes of the hippocampus activated, and these upregulations of pro-inflammatory-related molecules were inhibited by LEV treatment. These findings suggest that LEV is likely involved in neuroprotection via anti-angiogenesis and anti-inflammatory activities against BBB dysfunction in the acute phase of epileptogenesis after SE.

Introduction

Levetiracetam (LEV) is an established second-generation anti-epileptic drug (AED) that exerts broad-spectrum anti-epileptic effects and is widely used to treat partial onset and generalized seizures (Lyseng-Williamson, 2011). In addition, LEV is a candidate second-line AED for status epilepticus (SE) (Manno, 2011, Glauser et al., 2016) and a candidate anti-epileptogenic drug (Pearl et al., 2013, Klein et al., 2012). One of the pharmacological mechanism unique to LEV is its ability to bind to SV2A, a protein of the synaptic vesicle complex, to inhibit neurotransmitter release (Lynch et al., 2004, Meehan et al., 2012). In addition, several other mechanisms of LEV have been reported concerning the control of neurotransmitter release (Cataldi et al., 2005; Nagarkatti et al., 2008, Rigo et al., 2002; Lukyanetz et al., 2002).

Animal studies have shown that LEV exerts anti-epileptogenic and neuroprotective effects for the treatment of a pilocarpine (PILO)-SE model (Mazarati et al., 2004, Zheng et al., 2010, Itoh et al., 2015). However, the findings from previous reports in other post-SE animal models have been conflicting regarding whether LEV can prevent or modify epileptogenesis (Löscher et al., 1998, Glien et al., 2002, Klitgaard and Pitkanen, 2003, Stratton et al., 2003, Gibbs et al., 2006). Furthermore, the mechanisms responsible for the anti-epileptogenic and neuroprotective effects of LEV are still unknown.

Post-brain insult epilepsy (post-traumatic, PTE; post-stroke, PSE; and post-SE, PSEE; etc.) accounts for approximately 20% of symptomatic seizures and 5% of all epileptic seizures (Herman, 2002, Brodie et al., 2009). Given such prevalence, the prevention of these post-brain insult epilepsies is one of important issue. However, although 47 clinical studies have examined the efficacy of conventional AEDs (e.g. phenobarbital, valproate, carbamazepine, phenytoin, lamotrigine, topiramate), none of these drugs was able to prevent the development of epilepsy (Temkin, 2001, Temkin, 2003, Temkin, 2009, Krumholz et al., 2015). Therefore, several non-AEDs, such as anti-inflammatory drugs and mTOR inhibitors, were recently examined in basic and clinical studies to prevent these acquired epilepsies (Galanopoulou et al., 2012, Jiang et al., 2012). While LEV is an AED for managing post-brain insult epilepsies, several recent clinical studies for LEV in PTE, PSE, and PSEE have suggested that it may decrease the risk of acquired epilepsy or prevent the development of epilepsy, where conventional AEDs failed (Belcastro et al., 2008, Klein et al., 2012, Pearl et al., 2013). LEV has promising pharmacokinetic properties, including excellent bioavailability (>90%), linear kinetics, low plasma albumin binding (<10%), and a rapid rate of reaching steady state concentrations. In addition, LEV does not have any drug-drug interactions with AEDs or other agents that operate via the hepatic CYP-dependent metabolic pathway (Cloyd and Remmel, 2000, Patsalos, 2000, Panayiotopoulos, 2010).

We focused our attention on preventing the development of post-SE epilepsy. SE causes 3–5% of cases of symptomatic epilepsy; as such, SE patients are at a high risk of developing acquired epilepsy (Hesdorffer et al., 1998, Temkin, 2003, Jacobs et al., 2009). Various clinical trials have indicated that conventional AEDs suppressed acute seizures, but so far, none have been able to prevent the development of post-SE epilepsy ( Temkin, 2001, Temkin, 2003, Temkin, 2009). Although the mechanisms underlying the relationship between SE and the development of epilepsy as part of the epileptogenic process are not well understood, the lack of efficacy of the conventional AEDs suggests that the biological mechanisms of the epileptogenic process may be differ markedly from that of the established epileptic brain models (Pitkanen et al., 2009).

Therefore, in the present study, we used a mouse model of PILO-induced SE as a model of epileptogenesis and investigated whether or not LEV treatment could protect against the SE-induced BBB failure associated with angiogenesis and brain inflammation in the latent period after SE.

Section snippets

PILO-induced SE developed brain edema in MR images

In the MRI study, the SI of T2WI and DWI was measured in epileptogenic brains during the latent period after SE termination by DZP (Fig. 1A). At 2 days but not at 3 h post-SE, T2WI signal hyperintense areas were identified in the limbic regions (dorsal hippocampus, amygdala and piriform cortex) (Fig. 1A, b and c). In contrast, increased SI of DWI compared to the findings in pre-SE animals (Fig. 1A, e) was observed in the dorsal hippocampus and amygdala and piriform cortex at both 3 h and 2 days

Discussion

The ZP prevented BBB failure associated with angiogenesis and neurodegeneration induced by inflammatory responses in PILO-SE model mice.

LEV, one of the newer AEDs, has a unique mechanism of action, wide therapeutic spectrum, and a favorable pharmacokinetic profile (Panayiotopoulos, 2010). In addition, this drug may also prevent or modify the development of acquired epilepsy in basic and clinical studies (Löscher et al., 1998, Klitgaard and Pitkanen, 2003, Belcastro et al., 2008, Klein et al.,

Experimental animals

The protocols for all animal experiments were approved by the Tokushima Bunri University Animal Care Committees and were performed in accordance with the National Institutes of Health (USA) Animal Care and Use Protocol. All efforts were made to minimize the number of animals used and their suffering. Male, eight-week-old ICR mice were purchased from Japan SLC (Shizuoka, Japan). All mice were maintained with laboratory chow and water ad libitum on a 12-h light/dark cycle. The utilized animals

Conflicts of interest

The authors declare that there are no potential conflicts of interest related to the present manuscript.

Acknowledgements

This work was supported by JSPS KAKENHI Grant number JP16K10216 (to K.I.), JP15K18947 (to R.K.) JP15K08122 (to H.N.) and JP26740024 (to Y. I.) and was financially supported in part by Tokushima Bunri University. This manuscript has been checked by a professional language editing service (Japan Medical Communication, Inc).

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    These authors contributed equally to this work.

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