Using our technique, we can test potential anti-epileptic substances within human brain samples to aid in drug development for temporal lobe epilepsy. Human brain tissue has the advantage of breaching the translational cap between basic research and clinical applications. On the day of the procedure or as late as possible on the day before, thaw 50 milliliters of a 10x choline aCSF solution in a 37 degrees Celsius water bath and add the thawed solution and 50 milliliters of 10x solution two to approximately 300 milliliters of double-distilled water.
Add final concentrations of glucose and calcium chloride to the mixture and stir until dissolved. Then, add double-distilled water to a final volume of 500 milliliters. Measure the osmolarity.
And fill a bottle with approximately 100 milliliters of the 1x choline aCSF. On the day of the procedure, chill the 1x choline aCSF on ice and use a glass gas dispenser connected to carbogen gas to carbogenate the solution for at least 10 to 15 minutes. At least 10 to 15 minutes before the procedure, prewarm the storage aCSF, high potassium plus 4-aminopyridine aCSF, and low magnesium plus bicuculline to 35 degrees Celsius with carbogenation.
To prepare the interface chamber, cut two four-by-two-centimeter pieces of filter paper for each slice-holding compartment and stack the pieces on top of one another. Then, place thin cotton strings around the pieces of filter paper inside the compartments to break the tension of the solution and to ensure an even flow. And place three to four 1.5-by-one-centimeter pieces of filter paper on top of the larger piece of filter paper in each compartment.
Before acquiring the tissue slices, use 70%ethanol to wipe the preparation area and place a cover over the area. Place super glue, two sharp tweezers, a spatula, a scalpel with blades, and a blade for rough cutting of the brain tissue near the vibratome. Wipe the buffer tray and specimen plate of the vibratome with 70%ethanol.
When the buffer tray is fully dry, cover it with aluminum foil and place the tray in the ice bath. Fill the ice bath with crushed ice and maintain the bath at minus 20 degrees Celsius until preparation. Then, wipe the vibratome and razor blade with 70%ethanol and calibrate the vibratome to minimize any vertical vibrations and tissue damage during the slicing procedure.
Immediately after acquiring the tissue sample, remove the tissue from the transport choline aCSF and cut away any burned portions of tissue. Cut an even surface to facilitate gluing of the tissue piece onto the specimen plate and use the vibratome to slice the brain tissue into 400-micrometer-thick slices, adjusting the amplitude and speed of the vibratome as necessary during the cutting. Some parts of the human hippocampus can be more resistant to cutting than others.
Taking extra care and time can greatly improve sample quality. Use a scalpel to trim the brain slices to fit into the recording chamber and use a spatula and small forceps to carefully place the slices onto the small pieces of filter paper in the interface chamber for at least one hour. For epileptiform activity recording, place a semi-permeable membrane glued to a plastic ring into the chamber and connect the inflow and outflow tubes of the chamber to a peristaltic pump.
Fill the tubes and chamber with pre-warmed carbogenated aCSF. To prepare the recording electrodes, fill pulled one to two megaohm glass pipettes with 154 millimolar sodium chloride solution. Place the pipettes into an electrode holder and use tweezers and a spatula to transfer a hippocampal slice from the interface chamber into a Petri dish filled with carbogenated aCSF.
Remove the filter paper without flipping the slice and place the slice into the recording chamber. Hold the slice in place with slice mesh and place the electrodes in the region of interest. Begin the recording.
The burst activity induced by the high potassium plus 4-aminopyridine should be visible two to five minutes after wash in, while the induction of seizure-like events by low magnesium plus bicuculline can take up to 30 minutes. The application of high potassium plus 4-aminopyridine induces epileptiform activity in the form of burst events within a few minutes. Seizure-like events with a duration of over 10 seconds can be induced with the application of low magnesium plus bicuculline.
The number of burst events decreases both during the application of lacosamide and the application of dimethylethanolamine, although the amplitudes are mostly unaffected. The quality of the brain tissue can also be improved by reducing the contamination and damage during the preparation. Human brain slices prepared with our methods can also be used to investigate basic physiological functions of neurons using patch clamp or investigate gene expression using various techniques.
Labs all over the world have begun to study basic mechanisms within the human brain, and these insights can be used to aid in drug development and can be tested using our proposed methods.
