Effects Of An Astrocytic Gap Junction Blocker Meclofenamic Acid On Limbic Epileptogenesis In Mice Kindling Models

Epilepsy is a common chronic neurological diorders characterized by recurrent, unprovoked seizures that can lead to sudden brain disfucntion. Epileptogenesis, broadly defined as the transformation of the normal brain to a chronic epileptic state, bears complex cascades of changes in genetic, molecular, cellular and circuit levels. Currently, most antiepileptic drugs (AEDs) available are only symptomatic treatments that suppress seizures and few of them have antiepileptogenic or disease-modifying properties that prevent or delay the development of epileptic seizures. Thus the most avid goal for antiepileptic drugs (AEDs) development today is to discover potential agents to prevent epilepsy or slow the process of epileptogenesis.Accumulating evidence reveals that gap junctions in the brain may be involved in epileptogenesis. However, Meclofenamic acid (MFA), an astrocytic gap junction blocker (primarily through connexin43), has not yet been applied in epileptogenic models to test whether it has antiepileptogenic or disease-modifying properties or not. In this study, we sought to investigate the effects of MFA on limbic epileptogenesis in amygdaloid kindling and hippocampus rapid kindling models in mice. We also tested the effects of MFA in Pentylenetetrazole (PTZ)-induced seizures in mice. In addition, we measured the changes of Cx43, the main connexin type in astrocytes, during hippocampus rapid kindling progression and investigated the effects of the gap junction general opener Trimethylamine (TMA) in amygdaloid kindling acquisition in mice. We found that intracerebroventricular (i.c.v.,2μl) administration of either dose of MFA (100μM,1mM or100mM)15min prior daily kindling stimulus decreased seizure stage, shortened the after-discharge duration (ADD) and increased the number of stimulations required to elicit stage5seizure. MFA also prevented the establishment of post-kindling enhanced amygdala excitability, evident as the increase of afterdischarge threshold (ADT) compared with pre-kindling values. Interestingly, MFA (100mM) had an acute effect of elevating the ADT in naive mice, while lower dose of MFA (100μM or1mM) failed to interfere with the ADT. But, MFA (100mM) did not affect the power spectra of amygdaloid EEG. Furthermore, MFA also retarded kindling acquisition in mice hippocampus rapid kindling model by prohibiting the progression of seizure stage and ADD, increasing the number of stimulations required to elicit stage5, increasing the number of stimulations stayed in stage1-2(focal seizures) as well as decreasing the number of stages stayed in stage3-5(generalized seizures). This data demonstrated that the antiepileptogenic effects of MFA were not specific to the amygdala but also occur in other limbic structures such as the hippocampus. Suprisingly, we found that MFA (1mM and100mM, i.c.v.) enhanced the seizure susceptibility in PTZ induced epilepsy models, evident as the shortened time latency to stage3or stage4and the increased percentage of tonic-clonic seizures. Although blocking astrocytic gap junctions by MFA can significantly retarded the limbic epileptogenesis, both hippocampus and cortex Cx43remained slightly regulated or unchaged during hippocampus rapid kindling progression. Moreover, generally opening all the gap junctions including those connecting neurons and astrocytes by TMA, did not exhibited pro-epileptogenic effect as predicted. Instead, TMA (10mM) slowed the progression of seizure stage, shortened the ADD and required more electrical stimulations to elicit stage5.Our results confirm that, although MFA seems to have pro-convulsant effects in PTZ-induced seizures, it can slow the limbic epileptogenesis in both amygdaloid kindling and hippocampus rapid kindling models, and indicate that as an astrocytic gap junction blocker, MFA may be a potential drug that has antiepileptogenic or disease-modifying properties. This oberservation also provokes a novel thought that proconvulsant drugs could have favourable effects on epileptogenesis, resulting in the proposal of pursuing proconvulsant prophylaxis for epileptogenesis. Note that both hippocampus and cortex Cx43remained slightly regulated or unchaged during hippocampus rapid kindling progression, we still would anticipate that the function of astrocytic gap junction may be changed during epileptogenesis. Indeed, we have proved that either blocking or opening the gap junction by pharmacological manupilations resulted in interrupting the epileptogenesis in mice kindling models. However, the drawbacks of manipulation with pharmacological agents are their low selectivity, off-target effects, and even activation of independent mechanisms that interfere with seizure susceptibility rather than the gap junction itself. Therefore, the interpretation of these results is currently problematic. More selective pharmacological manipulations and conditional gene knockout techniques should be generated and used in further investigating the mechanisms of the antiepileptogenic effects of both MFA and TMA, as well as the role of astrocytic gap junctions in epileptogenesis.