Electrophoretic Delivery of γ-aminobutyric Acid (GABA) into Epileptic Focus Prevents Seizures in Mice

Epilepsy is the fourth most common neurological disorder: about 1% of the population suffers from epilepsy, and about one-third of the affected have recurrent seizures. In most cases, seizures can be controlled with medication. However, drug treatment needs to be set for every patient individually, where proper dosing can take years to find^1,2. Additionally, most of the medication has serious side effects that reduce the quality of life^3,4,5,6,7. Finally, in 30% of the cases patients are resistant to medication, and in case of a constant single seizure generator locus, only resective neurosurgery can attenuate the occurrence of seizures⁸. Therefore, a major initiative in modern epilepsy research is to discover new strategies which can prevent recurrent seizures in patients at risk, while reducing the necessity of strong drug therapies and invasive resective surgeries.

Epileptic seizures occur when there is an imbalance within excitatory and inhibitory circuits either throughout the brain (generalized epilepsy) or in a localized part of the brain (focal epilepsy), such that neurons discharge in an abnormal fashion^9,10,11. Antiepileptic drugs can act in two different ways in seizure prevention: either decreasing excitation or enhancing inhibition¹². Specifically, they can either modify the electrical activity of neuronal cells by affecting ion channels in the cell membrane¹³ or act on chemical transmission between neurons by affecting the inhibitory neurotransmitter GABA or the excitatory glutamate in the synapses^14,15. For some medications, the mode of action is unknown¹⁸. Also, drug treatments have a continuous effect on patients and cannot adapt to the prevalence dynamics of seizures. Ideally, drugs with specific mechanisms of action would act on the underlying epileptic processes. An optimal treatment would not touch the brain interictally but would act immediately when a seizure starts developing. In contrast to that, in all cases of epilepsy, medication now means a systematic treatment, affecting the whole brain and the whole body of the patient⁹.

Epileptic seizures can appear many years after the initial insult such as brain trauma. The period between the initial insult and the occurrence of the first spontaneous seizures is characterized by considerable molecular and cellular reorganizations, including neuronal death with the disappearance of neuronal network connections and axonal sprouting/neosynaptogenesis with the appearance of new connections^19,20,21. Once seizures become recurrent, their frequency and severity tend to increase, involving more brain regions. It is important to distinguish the sites of seizure onset (epileptogenic regions) from propagation networks, as the rules of seizure genesis and propagation may differ. Research performed on human tissue and experimental models of epilepsy have provided important data regarding the reorganization of circuits and their ability to generate seizures^20,21,22,23. However, it is difficult to determine if these reorganizations are adaptive responses or whether they are causally related to epileptogenesis or seizure genesis and propagation¹².

Therefore, localizing the epileptic focus and applying antiepileptic drugs locally are one of the main challenges in contemporary epilepsy research. Several experiments using animal models of epilepsy and some clinical studies aimed to find the onset of the seizure events and define the underlying mechanisms in the brain^24,25,26,27. To this end, we developed a new experimental protocol using the 4AP-induced epilepsy model^28,29,30,31 in an acute mouse preparation, which allows the precise insertion of three devices into the given area of the hippocampus, where network activity in vivo is manipulated in a highly localized manner. Localized 4AP injection by a glass micropipette helps to induce epileptic SLEs in a localized spot in the hippocampus, while with the help of the novel polymer-based µFIP probe the control of the seizure activity is achieved simultaneously by recording the neuronal electrical activity with the device’s recording sites. Hippocampal local field activity is also monitored with a multichannel silicon probe in a layer-specific manner in the cortex and in the hippocampus simultaneously.

The recently invented µFIP probes work by using an applied electric field to push charged drugs stored in a microfluidic channel across an ion exchange membrane (IEM) and out to the surrounding tissue (Figure 1). The IEM selectively transports only one type of ion (cation or anion) and, thus, works to limit both passive diffusion in the “off” state and transport of oppositely charged species from the surrounding tissue into the device. The electric field is created on demand by applying a small voltage (<1 V) between the source electrode which is internal to the microfluidic channel and a target electrode which is external to the device (in this case, the head screw on the animal model). The rate of drug delivery is proportional to the applied voltage and the measured current between the source and target electrodes. The precise tunability of drug delivery is one of the primary advantages of the µFIP. Another critical advantage, compared to fluidic or pressure-based drug delivery systems, is that in the µFIP there is only a negligible pressure increase at the drug delivery outlet as drugs are delivered across the IEM without their carrier solution.

There is a small amount of passive leaking of GABA when the µFIP is “off”, but this was found not to effect SLEs. The µFIP are custom-made following conventional microfabrication methods that we reported previously³¹.

Since one way of preventing recurrent seizures is the blockade of network discharges at the very beginning or even before the first seizure event, the presented method for delivering the inhibitory neurotransmitter GABA into the epileptic focus has great therapeutic potential for seizure control in patients with focal epilepsy. Since GABA is an endogenous substrate, it leaves intrinsic neuronal properties unchanged in physiological concentrations. The local application of low levels of GABA will only affect cells naturally responsive to inhibition, and will only cause similar effects to physiological inhibition, contrary to deep brain stimulation (DBS), which has unspecific actions by stimulating all cells of the neuronal network in its environment, causing a mixed response involving both excitation and inhibition. In conclusion, the proposed method provides a more specific approach to seizure control than DBS.