Multi-electrode array Opnamen van Human Epileptische Postoperatieve Cortical Tissue

The overall goal of this procedure is to perform multi electrode array recordings of ex vivo, interictal, and seizure-like events from human epileptic cortical tissue, thus providing a way to address the basic mechanisms underlying epileptic activities, initiation, propagation, and the effects of anti-epileptic drugs at both the cellular and network levels. This is accomplished by first, carefully resecting the human cortical tissue from patients with pharmaco resistant epilepsy during surgery. The second step is to remove the blood clots, vessels, and meninges and the excess white matter before slicing.

Next, cut the block of tissue into 400 microns slices. The final step is to keep the slices in the interface conditions of high oxygenation and controlled temperature with slow perfusion. Ultimately, the multi electrode array technique is used to record spontaneous interictal like activity and evoked ictal like events.

Our laboratory investigates the role of neuroglial interactions in cerebral physiopathology to study how neural cells can generate pathological network activity in humans. We have recently established multi electrode array recordings of human epileptic, postoperative cortical tissue in collaboration with neck care and lap Petri hospitals. Today, Thomas Blo neurosurgeon at Neck Care Hospital, together with Feld epileptologist and researcher at Lapis Hospital and Elena DOI postdoc in my laboratory will demonstrate you how to perform and make good use of such techniques.

From the collection of human epileptic tissue to MEA recordings ex vivo, The use of human cortical tissue ECT to cure epileptic patients allows direct exploration of functionally maintained human epileptic neurons and glial cells, which are interconnected in specific networks. In vitro investigations, a human tissue give access to single cells and network activities, ionic based mechanisms, which cannot be fully addressed in vivo. MEA recording of human cortical tissue can help answer a key question in the epilepsy field, such as the cellular mechanisms underlying exogenesis and of seizure propagation.

The implication of this technique extend towards lesional epilepsy and their therapy because it allows to understand the mechanisms of the pharmaco resistance and the effect of the anti-epileptic drug on seizure-like event recorded in the human cortical slices. In vitro. The main advantage of multi electro arrays over existing methods like e, e, g, or in vivo micro electrodes, is that they allow the non-invasive, simultaneous stimulation and recording of field potentials and multiunit activity from several sides of the tissue.

To begin the procedure, prepare the interface chamber by first filling the lower section with distilled water. Then connect the chamber to a carbogen source. Next, cut a piece of lens tissue to fit the flat area of the slice chamber.

Place it next to the input tubing in order to allow bubbles to escape from the input line before reaching the slices. After that, cut two pieces of stranded cotton thread with the same length as the piece of lens tissue. Place them on the sides of the flat area of the chamber, set the flow rate of the peristaltic pump to one milliliter per minute.

Subsequently turn on the oxygenation for the water at the bottom part of the chamber. Then set the temperature controller to 37 degrees Celsius. Cover the upper part of the interface chamber with a piece of paraform and wait at least 30 minutes for the temperature to increase and stabilize.

Now, prepare the dissection tools, which include two fine forceps, a spatula, a small spoon, and a blade, two glass Petri dishes, two brushes, and the cyanoacrylate glue. Next, prepare the vibrato by setting the parameters for slicing. In this procedure, remove the prepared sucrose based A CSF from the minus 80 degrees Celsius freezer and crush it into slush.

Oxygenate it with carbogen. Then transfer 250 milliliters of the A CSF slush to a glass bottle and keep it in a doer bottle filled with ice for tissue collection in the surgery room. Under standard general anesthesia, use a neuronavigation system to allow a precise localization of the cortex.

Afterward, make an incision in the skin, then create a bur hole in the bone, followed by a craniotomy. Next, gently elevate the bone flap and open the dura with scissors. Subsequently perform on block resection of the epileptic cortex and avoid extensive coagulation.

Cut a one centimeter thick cortical block and place it into the frozen oxygenated sucrose based A CSF. Afterward, transfer the tissue in frozen sucrose based A CSF to the lab as fast as possible, but avoiding mechanical shocks. Now, fill a Petri dish and the vibrato buffer tray with the sucrose based oxygenated A CSF slush and continue to oxygenate the A CSF slush with carbogen.

Then carefully take the tissue out from the bottle with a spoon and place it in the Petri dish. Using the forceps carefully remove the blood clots, vessels, and meninges to avoid resistance during slicing. Cut side of the tissue with a blade to obtain a flat surface as parallel as possible to the opposite side of the tissue.

Then glue the tissue onto the vibrato specimen plate in order to prepare transverse cortical slices. Subsequently, place it in the buffer tray and cut 400 micron thick slices at low speed. Next, cut a few pieces of lens tissue with a spatula and a brush.

Transfer these slices of lens tissue to the interface chamber. Incubate them at 37 degrees Celsius for at least one hour before recording. In this procedure, plug the perfusion system into a peristaltic pump and the MEA system into a computer.

Then place an empty MEA chip inside the amplifier. Next, set the temperature controller for heating the cannula element in the MEA chamber to 37 degrees Celsius. Start the perfusion of the oxygenated A CSF at five to six milliliters per minute.

After that, open the recording software. Make sure all the MEA electrodes are well connected and that there is no noise due to the perfusion. Now pour some warmed recording A CSF in a glass petri dish.

Transfer a slice from the interface chamber to the Petri dish by grabbing the lens tissue. Then carefully detach the slice from the tissue by submerging it in the solution, or use a small brush to facilitate the detachment. Next, fill the MEA chip with some A CSF.

Transfer the slice to the MEA chip with a spatula with a plastic past pipette. Gently remove the solution to make the slice adhere on the electrode area and keep it in place with a platinum anchor. After that, add one milliliter of recording a CSF to the MEA chip and place it in the amplifier.

Then start the perfusion at five to six milliliters per minute. Next, check the slice position in the MEA recording area. By using the MEA monitor software, adjust it if necessary.

In order to record from the cortical layers and take a picture, start the recording. This is a representative trace of human cortical slice activity recorded by an MEA electrode. In normal A CSF, it is possible to observe interictal like spontaneous activity 15 minutes after the perfusion with magnesium free six millimolar potassium A CSF.

The first seizure appears and is followed by other events at two to three minute intervals. The blue trace illustrates the activity zoomed in. This panel shows the data high pass filtered at 250 hertz, which reveals multi-unit activity when zoomed in on here is a representative recording of a seizure from all MEA electrodes in magnesium free.

Six millimolar potassium A CSF. Each square represents an electrode of 12 by 12 MEA array and shows an 82nd time window. The doted line indicates the position of the cortical surface relative to the MEA array.

After watching this video, you should have a good understanding of how to safely transport human cortical tissue, prepare and store viable cortico psoriasis, and perform MEA recordings of spontaneous and induced epileptic activities While attempting this procedure, it's important to have a close collaboration between the clinical team in the hospital and the researchers in the lab. Never forget that working with human tissue can be hazardous, and protection should always be undertaken, such as wearing gloves in order to avoid any possible infectious disease Following this procedure. Other techniques such as fluorescence imaging, patch lamp, recording, spike sorting and histological analysis can be coupled to em EA recordings to correlate synaptic properties, single cell behavior ion dynamics and cell and molecular abnormalities to population activities.

Performing MEA recordings of human epileptic tissues can pave the way for researcher to explore lesional epilepsies in order to clarify the mechanisms underlying exogenesis and pharmaco resistance, as well as the role of neuroglial interaction in this phenomena. Thank you for watching and good luck with your experiment.