Epilepsy is a prevalent neurological disease. Rhinal cortex-hippocampus organotypic slices depict evolving epileptic-like events that resemble in vivo epilepsy and can therefore be used as an ex vivo model of epileptogenesis. This system is indeed an excellent tool for monitoring the dynamics and progression of epileptogenesis, as well as for screening potential therapeutic targets for this brain pathology.

Demonstrating the procedures with me will be my students Francisco Meda and Mafalda Carvalho. To harvest the brain from a postnatal day six to seven Sprague-Dawley pup, first wash the head three times in five milliliters of GBSS. Working in a biosafety cabinet, firmly insert sharp forceps into the eye sockets to hold the head in place and use thin tip scissors to cut the scalp along the midline from the vertebral foramen to the frontal lobes.

Cut the skull in the same manner and along the cerebral transverse fissure and use curved long forceps to move the skull pieces apart. Use a spatula to discard the olfactory bulbs and to transfer the brain dorsal side up into a 60 millimeter plate containing five milliliters of cold GBSS. Insert fine forceps into the cerebellum and insert the spatula along the midline to carefully open the hemisphere.

Use short curved forceps to carefully remove the excess tissue that covers the hippocampus without touching the hippocampal structure, then cut below each hippocampus. Transfer one hemisphere at a time hippocampus side up and parallel to each other onto a piece of filter paper. Place the filter paper onto a tissue chopper with the hemispheres perpendicular to the blade and cut the hemispheres into 350 micron slices.

Transfer the sliced tissue into a new Petri dish containing five milliliters of cold GBSS and use round tip electrodes to carefully separate the slices keeping only the samples with a structurally intact rhinal cortex and hippocampus. The DG and CA areas should be perfectly defined as well as the entorhinal and perirhinal cortex regions. Use a spatula and a round tip electrode to place up to four intact slices onto individual inserts in the appropriate number of wells of a six-well plate containing 1.1 milliliters of culture medium per well.

Use a P20 pipette to remove any excess dissection medium from around each slice. Place the plate in the cell culture incubator. Change the medium every two to three days.

Use forceps to lift the insert. Aspirate the medium from the corresponding well and place the insert back in place. Use a P1000 pipette to add one milliliter of fresh 37 degree Celsius medium to each well.

Prepare the electrophysiology setup in a closed circuit. Verify that the flow rate of the interface type chamber is set to two milliliters per minute and open the carb-ox valve. Check the water level in the system and place a piece of filter paper into the interface recording chamber to drain any excess medium.

Place a piece of lens cleaning paper beneath the frame to supply medium to the slice and turn on the temperature controller, amplifiers, and micro manipulators. Use a syringe to load a glass electrode with freshly prepared artificial cerebrospinal fluid. Place the glass electrode into the receiving electrode and wait for the temperature in the interface chamber to stabilize at 37 degrees Celsius.

Working in a biosafety cabinet, place the insert in a 60 millimeter plate with a drop of medium. Use a sharp blade to cut a slice from the insert and place the slice in the interface chamber with the hippocampus to the bottom right, then place the receiving electrode into the CA3 pyramidal cell layer, initiate the continuous acquisition protocol, and record the slice for 30 minutes. To perform the propidium iodide uptake assay, work in a biosafety cabinet.

Lift the insert from the well and add propidium iodide in medium to a final concentration of two micromolar per well. Slowly agitate the plate before placing the insert back into the well, taking care that there are no bubbles beneath the slices. When all slices have been treated, return the plate to the cell culture incubator.

For immunostaining of the slices, at the end of the propidium iodide incubation, aspirate the medium from the bottom of each well and add one milliliter of 4%paraformaldehyde to the top and bottom of each insert. After one hour at room temperature, wash the slices two times for 10 minutes and one milliliter of PBS per wash and use a hydrophobic pen to draw two rectangles on microscope slides. Use a sharp blade to cut the slices from the inserts and place one slice into each rectangle.

Add permeabilization blocking solution onto each slice for a three-hour incubation at room temperature. At the end of the incubation, add the primary antibodies of interest to each slice for a four degree Celsius incubation overnight. The next morning, wash the slices with PBS-Tween three times for 10 minutes per wash and incubate the slices with the appropriate secondary antibodies for four hours at room temperature.

Afterwards, wash the slices as demonstrated and add 50 microliters of Hoechst solution to each slice for a 20-minute incubation at room temperature. Following Hoechst incubation, wash the slices again and add 50 microliters of mounting medium to each one, then place a coverslip over the slices and seal with nail polish. After allowing the slides to dry for 24 hours at room temperature, visualize the immunostaining and propidium iodide uptake by confocal microscopy.

Rhinal cortex-hippocampus organotypic slices depict mixed interictal and ictal-like activity at seven days in vitro. At 14 days in vitro, spontaneous activity is characterized by ictal discharges, which evolve to an overwhelming ictal activity at 21 days in vitro with ictal events lasting greater than one minute. Propidium iodide uptake and immunohistochemistry against the neuronal marker NeuN revealed some neuronal death in seven days in vitro slices that is considerably increased at 14 days in vitro in all areas of the hippocampus.

At seven days in vitro, ramified microglia with a low CD68 expression are more abundant than Iba1 positive CD68 positive reactive microglia. Whereas at 14 days in vitro, in all areas of the hippocampus, Iba1 positive CD68 positive amoeboid M1 microglia exceed microglia with a low CD68 expression. At 14 days in vitro, some Iba1 positive CD68 positive cells with a hyper ramification appearance can be pinpointed, suggesting the possibility of an M2 anti-inflammatory microglia phenotype.

At seven days in vitro, the expression of CD3 is barely detectable, while in 14 day in vitro slices, hypertrophic glial fibrillary acidic protein positive CD3 positive astrocytes can be observed, suggesting a progressive activation of A1 astrocytes. Prepare rhinal cortex-hippocampus slices with extreme care. Make sure to remove the excess tissue above the hippocampus as it can compromise slice integrity during slicing.

Structural deficit will negatively impact upon slice viability. In addition to the demonstrated approaches, a plethora of assays such as Western blot analysis, real-time PCR and ELISA can be applied to this system to address specific questions regarding epileptogenesis.

Summary

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Organotypic Slice Cultures