Fabrication of an Expandable Brain Matrix Customizable Across Developmental Stages

Fabrication of an Expandable Brain Matrix Customizable Across Developmental Stages, by Chapman and colleagues at the University of Kentucky in Lexington, Kentucky. The development of the piglet brain is very similar to that of a human brain in several aspects. Compared to rats and mice, the ratio of piglet brain weight from birth to maturity is much closer to humans and the functional regions of piglet and human gyri and cephalic brains are very similar.

For these reasons, the use of pigs and other large mammals in neuroscience research is increasing. However, commercial brain matrices for larger mammals are expensive, and choices are often limited to a single size or plane of sectioning. Consequently, commercial matrices may not provide a good fit for different strains, both males and females, and different ages.

This is particularly problematic for developmental neuroscience studies, in which brain sizes change markedly with postnatal growth. For example, piglet brains grow quickly during the first three months, increasing in volume by more than threefold. The commercial piglet brain matrix is designed to fit brains from piglets only up to two weeks of age.

Here we present an acrylic plate-based customizable matrix building method as a cost-effective and easy-to-implement solution to this problem. Four naive four-day-old pigs were obtained for this experiment. One was utilized for brain collection at five days of age, two at 32 days of age, and one at 38 days of age.

After removal of the brain from the skull, the brain was immersion fixed in 10%buffered formalin for two days, and placed in a 30%sucrose solution for five days for cryo-preservation. We will next present how to make a cast of the brain using agarose gel. The first step to making the matrix is to make a mold of the brain.

We will illustrate the process for creating a mold of a postnatal day five piglet brain, which will be used to build a sagittal matrix. Place a strip of heavy duty plastic at the bottom of the glass container, making sure to leave both ends extending out of the container edges. This strip will make the extraction of the agarose block easier.

Submerge the container about halfway in a bucket of wet ice. In a glass beaker, heat 150 milliliters of PBS to 90 degrees centigrade and add six grams of agarose, stirring constantly. Slowly pour the 4%agarose solution to create a layer of a half to one centimeter thickness on the bottom of the container and allow it to solidify.

Completely coat the brain in a thin layer of mineral oil to prevent the agarose from binding to the brain surface or becoming trapped in the sulci. The oil also makes removal of the brain from the agarose gel much more manageable. Once coated, place the brain on the thin layer of agarose in the desired orientation for a coronal or sagittal mold as straight and upright as possible.

Allow the agarose solution to cool to 50 degrees Celsius while stirring constantly to retain a pourable viscosity. Then pour the agarose solution over the brain until it is completely embedded. Allow the agarose to solidify at room temperature.

Cut the agarose layer above the brain with a surgical probe by following the brain outline. Once the cutting is complete around the brain, use a thin spatula to gently lift off the top layer of the agarose. Take care not to slice or nick the brain during these steps.

Gently remove the whole brain from the mold. It should come out relatively easily because of the mineral oil that was applied to the surface earlier. Make sure to remove any small pieces of agarose gel that may have fallen into the impression.

Heat 98 milliliters of PBS in a beaker to 9 degrees Celsius and add four grams of agarose and two milliliters of India ink. Stir well. Pour this black agarose solution into the impression made by the brain, taking care not to overflow the impression.

Allow the black gel to solidify. Hold both ends of the plastic strip and lift the entire agarose block from the container. We will now illustrate how to create template plates for your matrix.

To slice the brain mold, place one piece of scale paper in between two 76 millimeter transparent acrylic plates and put them close to the edge of the table. Then use clamps to secure four corner squares and two acrylic plates in order to build two walls to hold the agarose blocks snugly in the desired orientation. The front edges of the two metal corner squares and the two vertical acrylic plates should be aligned along one side of the scale paper.

Place the agarose gel block so that the edge to be cut aligns along the scale paper and with the edges of the vertical acrylic plates and corner squares, and place a weight on the opposite side of the agarose block to hold it in position. Trim any excess off the block until the India ink brain cast is just about to be cut. Move the agarose block forward at desired steps, for example, two millimeters or five millimeters with the help of the markings on the underlying scale paper.

Grab the blade firmly with both hands and guide it down the front edges of both acrylic plates and corner squares to slice the block. Place each slice onto a second sheet of scale paper and press down firmly to transfer an impression onto the paper. With a pen, outline the entire slice.

Remove the slice and outline the brain contour identified by the India ink. Note the orientation of the first slice on the scale paper, for example, posterior and anterior for sagittal sections, and keep it consistent when additional gel slices are added to the paper throughout the process. Number each tracing sequentially.

Cut the acrylic plates to 10 by 8 1/2 centimeters. Then stack them and secure them to the stage of a drill press using corner squares and clamps. Drill three holes through the stack with the coordinates indicated in the manuscript.

Remove the protective film from the acrylic plates. Then place an acrylic plate above a template on the scale paper and align the top of the acrylic plate lines to the top edges of the template. Trace the outline of the brain onto the acrylic plate with a permanent marker.

Label each plate to match the number of the template or the brain tracing. Using the bandsaw, cut each plate along the copied template lines. Alternatively, a rotary tool with a wood carving tip can be used to remove unwanted areas.

Polish the cutting edge using a rotary tool with a polishing sanding tip to smooth the edges as sharp edges could damage the brain during slices. Recheck the accuracy of cutting by placing the acrylic plate over the template. Trim and polish as necessary.

Next, we will illustrate the assembly of the plates. Preassemble the matrix by stacking the plates in sequential order and check the alignment of the plates. Matrix assembly starts with placing three long stainless steel bolts through the first acrylic plate and then placing a washer on every screw.

Place the second plate and add washers to each screw. Alternatively, the washers can be glued around the anchoring holes with super glue to avoid having to add the washers individually during each assembly. Repeat this process until all the plates have been added sequentially to the bolts.

Tighten everything together with a nut on each bolt. Perform a final polishing of the edges facing the brain to make a smoother transition from plate to plate. Use different colored markers to color the top edge of the acrylic plates with alternating colors to make slicing much easier.

Here we alternated black, red, and unmarked plates. Finally, we will demonstrate the process for slicing the brain in either the coronal or sagittal plane. To slice the brain, place it in the matrix in the same orientation that it was during the molding process and as straight as possible.

You will need a wide, flat blade to cut the brain. The wide blade limits the potential for twisting or bending during the slicing. We recommend a high profile cryostat blade.

For safety, wrap about a two centimeter length of one end of the blade with lab tape to create a space to safely hold the wrapped end during slicing of the brain. A note of caution here. Since each part of the brain has different textures after formalin fixation, care should be used to apply even pressure on the blade.

Moreover, the piglet brain is large, so it is better to use a slicing motion to cut the brain than a direct, pushing down cutting. The blade should be pulled with constant force from one side to the other, making sure to go far enough down to slice through the entire brain. Put all the slices into a PBS-filled six well plate or stainless tray.

Carefully lay the slices into the tray, maintaining the slice order and orientation, and preserve or proceed as needed. To adjust the geometry or expand the cavity for a better fit for the brain, selected acrylic plates can be removed or added to adapt the matrix to different brain sizes. Here we illustrate the expansion of a sagittal matrix built for a P5 piglet brain by adding several plates, here colored blue and green, to the mid portion of the matrix.

The matrix is now suitable for sagittal sectioning of a P38 piglet. The ability to expand or reduce the size of the matrix greatly increases matrix flexibility and can save both materials and effort. As with the coronal slicing, carefully cut the brain in the sagittal plane using successive plates to guide the blade.

The color coding system helps identify the correct sequential placement of the blade, which is difficult when the slices are thin. Remove the brain from the matrix and gently tease apart the slices. In conclusion, we have developed an easy, cost-friendly, and flexible method to create brain matrices for neonatal piglets.

This concept can be applied to older pigs and to other large animal models.