Electroencephalography (EEG) is a non-invasive technique used to record electrical activity generated by neurons in the brain. This method involves placing electrodes on the scalp to measure voltage fluctuations resulting from ionic current flows within the neurons of the brain. EEG is a valuable tool for both clinical and research purposes, providing insights into brain function and aiding in the diagnosis of neurological disorders. It is widely used in the study of brain dynamics, cognitive processes, and the neural mechanisms underlying various mental states and behaviors.

In epileptic patients, EEG plays a crucial role in diagnosing and monitoring epilepsy. During resting state conditions, EEG can reveal abnormalities such as interictal spikes, which are indicative of epileptogenic regions even when the patient is not experiencing a seizure. Experimental setups are also used to identify the modulatory effects of vagus nerve stimulation (VNS). Techniques such as photic stimulation (flashing lights), hyperventilation, and specific cognitive or motor tasks can be employed to observe how VNS influences brain activity.

We account with a Biosemi EEG recording system, offering a range of configurations suitable for various research and clinical needs. This system supports low to mid-density electrode setups, such as configurations with 19, 21, or 64 electrodes, providing flexibility in spatial resolution. Additionally, the system allows for high temporal resolution with sampling rates of 1024, 2048, or 4096 Hz, ensuring detailed capture of rapid neural dynamics. This combination of spatial and temporal resolution makes the EEG system an excellent choice for detailed and accurate EEG recordings in both clinical and research settings.

SEEG is part of phase 2 evaluation performed in patients with refractory epilepsy in which the noninvasive work up was not able to clearly identify  the epileptogenic zone. SEEG is typically performed by a neurosurgeon at Clinique Universitaires Saint Luc (CUSL).

The procedure consists in introducing through the skull, multiple intracranial electrodes inside the brain parenchyma in order to record more accurately the electrophysiological activity of different regions of interest- selected after a multimodal epilepsy work-up. Due to its invasive nature, implying some limited surgical risks, these recordings are limited to highly selected epileptic patients.

The epilepsy and Neurostimulation Lab has direct acces to this patient population through collaboration with the Center of Refractory Epilepsy and the department of Neurosurgery of Cliniques Universitaires Saint-Luc

Functional Connectivity based on EEG is a technique used to study the interrelationships between different regions of the brain by analyzing the statistical dependencies of their electrical activity. This method involves recording brain waves through electrodes placed on the scalp and then applying various mathematical and computational approaches to assess how different brain regions interact with each other. Functional connectivity provides insights into the dynamic communication between neuronal populations and is crucial for understanding the brain’s functional organization. By examining these connections, researchers can explore how different parts of the brain coordinate and integrate information.

This technique is closely related to Network Graph Theory, a field of mathematics that studies the properties of networks through nodes (representing brain regions) and edges (representing connections between these regions). By modeling the brain’s functional connectivity as a graph, researchers can use metrics from graph theory, such as degree, centrality, and clustering coefficient, to quantify the brain’s connectivity patterns. In epileptic patients, analyzing functional connectivity can reveal abnormalities in brain networks, such as hyper-synchronization or disrupted connectivity, which are often associated with seizure activity. This approach helps map the connectome, the comprehensive map of neural connections, and understanding how epilepsy alters brain network dynamics.

Furthermore, functional connectivity analysis can provide valuable insights into how vagus nerve stimulation (VNS) modulates brain networks.

The Eyelink 1000 is a sophisticated eye-tracking device used to measure pupil size and gaze direction with high precision. To measure pupil size, the Eyelink 1000 uses infrared illumination and a high-resolution camera to capture detailed images of the eye. The device tracks the corneal reflection and the dark pupil to calculate the pupil diameter in real-time. The Eyelink 1000’s software analyzes these images, providing continuous and accurate measurements of pupil size. The high sampling rate and spatial resolution of the Eyelink 1000 make it an ideal tool for precise and reliable pupillometry.

The P300, also known as P3, is an event-related potential (ERP) component observed in electroencephalography (EEG) recordings. It is characterized by a positive deflection in voltage occurring approximately 300 milliseconds after the presentation of a stimulus. The P300 is considered an index of cognitive processing, particularly related to attention and the allocation of mental resources. An auditory oddball task is a common experimental paradigm used to elicit the P300 response, and the one used in our research projects. In this task, patients are presented with a series of auditory stimuli that include frequent « standard » tones and infrequent « oddball » tones. The subject’s task is to detect and respond to these oddball tones. The infrequent and unexpected nature of the oddball tones captures the subject’s attention and engages cognitive processes, leading to the generation of a P300 response. This response is most prominent at parietal electrode sites, such as Pz, but can also be detected at central and frontal sites like Cz and Fz.

Laryngeal responses can indicate VNS-induced A-fiber activation by recording surface laryngeal motor evoked potentials (LMEPs). These LMEPs act as markers of efferent A-fiber activation, particularly from the recurrent laryngeal nerve, a branch of the vagus nerve, and can be captured using two surface electrodes positioned at the neck surface.

MRI is a non-invasive and non-radiating imaging technique, allowing to obtain high-resolution images ranging from array of soft tissues to different anatomical region of the body such as the brain. Different types of MRI sequences exist and can be used depending on the process or structure to be studied. Our research uses a multimodal MRI approach to characterize the brain tissues of patients with epilepsy, encompassing both structural and functional acquisitions.

A structural MRI sequence aiming at visualizing the locus coeruleus – a small brainstem nucleus involved in the antiseizure effects of VNS – has been developed and optimized in-house in our lab with the SIGNA^TM Premier 3T MRI system of Saint-Luc University Hospital. Part of our research focuses on evaluating the integrity of this brainstem nucleus and its potential impact on the therapeutic effectiveness of VNS.

High-gradient multi-shell diffusion MRI was also used in our lab and constitutes a powerful imaging technique to evaluate white matter microstructure based on the diffusion of water molecules within the brain. In this context, our lab investigated how the integrity of various white matter tracts – including tracts projecting from the locus coeruleus to the hippocampus, and thalamocortical tracts – could be linked to the therapeutic effectiveness of VNS, using different (multi-compartment) diffusion models. These models included: Diffusion Tensor Imaging (DTI), Neurite Orientation Dispersion and Density Imaging (NODDI), distribution of anisotropic microstructural environments in diffusion-compartment imaging (DIAMOND), and Microstructure Fingerprinting (MF).

Functional MRI is used to measure and map brain activity in an array of clinical conditions. Using task-based paradigms, changes in cerebral blood flow occur in response to neural activity in specific brain regions involved in the task being performed.

Resting-state functional MRI is similar to the “classical” functional MRI in regard to its physical aspect (i.e. measuring the fluctuation of the blood flow in the different regions of the brain) but differs with the type of activity asked from the patient. The subject is asked to empty his mind and try to avoid thinking about anything, and the sequence records thus the activity of the brain at rest (hence the term resting-state). Thereafter, the signal is processed in order to identify the different regions that show a correlation between each other, depending on the purpose of the study.

An emerging approach to stimulate the vagus nerve non-invasively consists of the so-called transcutaneous auricular VNS (tVNS).  It is typically administered to the cymba conchae of the left ear.

EEG: Our custom-made epidural electrodes are engineered with a insulated wire, featuring a stainless-steel screw soldered at one end and a tip at the other, ensuring both reliable signal transmission and secure attachment for epidural recordings.

Depth EEG electrodes: The custom-designed depth EEG electrodes are constructed by twisting two coated stainless steel wires. The electrode is inserted with the aid of a stereotaxic frame to achieve the desired depth within the brain structure. Once correctly positioned, the electrode is fixated into a head cap which can be connected to our video-EEG-Vagus nerve activity recording recording system

Vagus Nerve Activity recording: The custom-constructed implanted tripolar micro cuff electrodes are designed for rat vagus nerve recoding purposes. The nerve is meticulously positioned within the cuff to optimize electrode contact, and the cuff is then sutured around the nerve to ensure a secure and stable fit.

Tissue Collection:

• • Transcardial Perfusion: This method involves perfusing the animal with paraformaldehyde through the heart to preserve the tissues and especially the brain. This ensures that the tissue samples are well-preserved and suitable for detailed analysis.
  • Vibratome Sectioning: After perfusion, brains are sectioned using a vibratome. This device allows to cut very thin, precise slices of tissue, which are essential for our molecular studies. Then they are conserved in -20°C in cryoprotectant before analysis.

Molecular Techniques:

• • • Immunohistochemistry (IHC): This technique involves using antibodies to detect specific proteins within the tissue sections. By applying fluorescent or chromogenic labels, we can visualize the distribution and abundance of these proteins under a microscope, providing insights into the molecular changes associated with epilepsy.
    • In Situ Hybridization (ISH): ISH is used to detect specific nucleic acid sequences within the tissue sections. This method allows to localize the expression of particular genes at the cellular level.
    • Quantitative Polymerase Chain Reaction (qPCR): qPCR is a powerful technique for quantifying the expression levels of specific genes. By measuring the amount of target DNA or RNA in our samples, we can assess how gene expression changes in response to epileptic conditions or treatments.

These techniques enable us to study the molecular and genetic underpinnings of epilepsy in detail, providing valuable insights that can lead to the development of new therapeutic strategies.

Photoplethysmography (PPG) is a non-invasive technique used to measure blood volume changes in the microvascular bed of tissue. This method involves using a light source and a photodetector to detect variations in light absorption, which correspond to the pulsatile nature of blood flow. The PPG signal primarily reflects the cardiac cycle, providing valuable information about heart rate and peripheral blood flow. In animal studies, particularly with rodents, PPG enables chronic, awake recordings that allow for continuous, non-invasive monitoring of physiological parameters such as oxygen saturation, heart rate, and breathing rate. This capability is essential for long-term studies without the need for invasive procedures.

The Mouse Ox System is a state-of-the-art PPG recording device tailored for use with rodents. This system offers high sensitivity and accuracy in detecting physiological signals, allowing for precise measurement of heart rate, breathing rate, and other vital parameters.

A custom-built setup for chronic electrophysiological and video recording includes a specialized cage equipped with low-noise amplifiers (LNA) and simultaneous recording capabilities for EEG, Vagus Nerve activity and video. This setup ensures high-fidelity recordings of both neural and behavioral data, providing a detailed and accurate representation of the animal’s physiological and neurological states. The LNAs minimize interference, enhancing the accuracy of the electrical signals captured from the brain and peripheral nerves. The ability to record EEG and VENG alongside video enables the study of brain-vagus nerve interactions and the correlation of neural data with observable behaviors. This comprehensive system is essential for advancing our understanding of the complex autonomic and neurological dynamics in epilepsy and other neurological disorders.