Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury. This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

The overall goal of this procedure is to apply cytotoxic stimulation to brain micro slices to create an in vitro stroke model. This is accomplished by first preparing micro slices from a freshly isolated brain. The second step is to equilibrate the micro slices with gentle agitation.

Once equilibrated, the micro slices are ready to receive a variety of experimental manipulations. Ultimately, results show that micro slices are viable for at least two hours post generation. The main advantage of our technique over other existing techniques, such as primary embryonic, neuronal cultures, is that our technique utilizes slices of mature brain tissue, which contain more than one cell type, and are therefore more representative of intact brain tissue.

The first step is to obtain fresh brain tissue. Dip the brain in a small amount of warm Krebs buffer. To remove any residual skin, fur or blood, prepare macle wain chopper with two sheets of filter paper moistened with warm Krebs buffer.

When ready, place the brain on the stage of the chopper. Keep the brain and filter paper moist with warm crebs buffer while working to prevent the brain from sliding. Avoid allowing too much liquid to accumulate on the surface.

Slice the brain into 250 micrometer coronal sections. Next, turn the stage 90 degrees and create 250 micrometer sagittal slices. After this step, the brain sections are referred to as micro slices.

Place the micro slices into a round bottom two with 10 to 15 milliliters of warm crebs buffer. Gently agitate the tube until individual micro slices are suspended, and then allow them to settle under gravity. Carefully remove the supernatant, which will be cloudy due to suspended cell debris, and add 10 milliliters of fresh warm crept buffer to the tube.

Repeat the washing procedure four times to remove the majority of the debris once thoroughly washed. Equilibrate the micro slices at 37 degrees Celsius with gentle shaking or inversion, and change the Krebs buffer every 15 minutes for one hour in total. After this time, add two milliliters of fresh 37 degrees Celsius Krebs buffer and transfer 15 to 20 micro slices to a flat bottom polystyrene five milliliter tube.

Using an extra wide bore pipette tip with smooth edges, spread the micro slices evenly over the base of the tube in a single layer so that each micro slice is not further than a few millimeters from the oxygenated atmosphere. Once properly arranged, incubate the micro slices in a 37 degrees Celsius water bath to provide proper oxygenation to the micro slices. Continually pass carbogen over the micros slice suspension with the gas line positions just above the surface to avoid mechanical disruption of the micro slices to direct contact with the gas For the best results.

It's critical that the micros slices be aerated correctly. It's important that the gas line be placed the right distance away from the surface of the microsite suspension. Several lines can be fused together to allow multiple stimulations to occur.

At the same time, a variety of stimuli can be used. In this example, the micro slices undergo AMPA stimulation. Treat the micro slices with an cytotoxic stimulus or Krebs buffer alone.

For a control on stimulated group. Remove the incubation solution at various times post stimulation and replace it with 300 microliters of ice cold homogenization buffer at the desired time point. Post stimulation, homogenize the micro slices on ice using a glass Teflon down homogenizer store the homogenous at minus 80 degrees Celsius until ready for further analysis.

Respiration rates show that micro slices collected from the chicken forebrain are viable for up to two hours post generation. The high A TP and low A DP levels found in micro slices are also characteristic of viable cells. In this example, tissue potassium content was measured and found to be just above background after dissection, but to have increased upon incubation with Krebs buffer, the addition of WABE and EGTA caused tissue potassium content to decline demonstrating that micro slices normally actively pump potassium and that levels are not due to potassium trapped in dead tissue or interstitial spaces.

Here micro slices were generated from the striatum and sensory cortex of male rats and depolarized using high potassium crebs buffer. The rate of glue N two B phosphorylation varied between the cortex and striatum, whereas the rate of glue a one phosphorylation was similar between the two areas. Once mastered, this technique can be completed in approximately two hours if performed correctly.