Fabrication of an Expandable Brain Matrix Customizable Across Developmental Stages

Summary

An innovative method to build expandable brain matrices to cut either coronal or sagittal slices is described for application to neonatal piglets. This budget-friendly approach utilizes acrylic plates carved using templates from agarose gel brain molding, applies to multiple species, and allows for expansion to accommodate brain growth.

Abstract

Increasing interest in using newborn piglets as a study model for neonatal development and pathology has raised the need for a brain matrix to accurately acquire histological sections. However, the fast growth of the newborn brain limits the utility of commercially available matrices to a narrow developmental window. Therefore, an adjustable brain matrix construction method was developed. First, a glass container was placed on wet ice, and a layer of 4% agarose gel was laid at the bottom. Next, a formalin-fixed brain was placed on this layer and embedded with 4% agarose gel. After the gel solidified, the gel above the brain was carefully removed, and the brain was extracted. The cavity was filled with 4% agarose gel with 2% Indian Ink. After solidification, the gel block was removed and sliced into the desired thickness (2 mm or 5 mm) and orientation (coronal or sagittal) to print the brain templates on scale paper. The templates were copied to a series of transparent acrylic plates (2 mm or 5 mm thickness) and cut out accordingly on a band saw. Finally, the plates were assembled and secured with three sets of bolts and nuts through predrilled alignment holes to build a customized brain matrix. Construction of both coronal and sagittal matrices is illustrated for a neonatal day 5 piglet brain with steps of 2 mm and a coronal matrix for postnatal day 32-38 brains with steps of 5 mm. By adding or removing proper acrylic plates, the matrix was adjusted to adapt to the various sizes of piglet brains. Thus, a simple, cost-effective method to customize brain matrices for varied sizes of large animals is introduced to the neurological research community.

Introduction

Piglet brain development closely mimics that of humans^1,2,3,4. Thus, there is increasing interest in using piglets for preclinical studies of neurodevelopmental disorders, neonatal and pediatric diseases, and injury (Figure 1A,B)^5,6,7,8,9,10. As seen in Figure 1, the neonatal piglet, as a large animal model, has a skull and brain size closer to that of a premature human fetus (Figure 1A,B), while the adult mouse and rat skulls are at least two magnitudes smaller.

However, there are challenges with the use of larger mammals in biomedical research as compared to the more widely used murine models. Neonatal piglet brains grow quickly during the first 3 months after birth. In the first 5 weeks of life, the piglet brain nearly doubles in weight and triples in volume (Figure 1C)^4,11,12,13. During the period spanning p5 to p38, brain growth and development of gyri and sulci were notable (Figure 1C). The weight and volume of the p5 piglet brain were 29.5 g and 18.9 mL, while the two p32 brains were 51.4 and 52.6 g, and 49.6 and 53.5 mL, respectively. At 38 days, the brain grew to 53.7 g and 52.5 mL, representing an 80% increase in weight and 170% increase in volume from p5. This rapid growth of the neonatal pig brain poses a challenge for accurate histological sectioning of brains from piglets of different ages, strains, or sexes, using a single matrix.

Commercial brain matrices for piglets have an approximate range in cost from $1,000 to $3,600 each and are only available in one size option, designed to be used for piglets that are 2 weeks old or younger. Although there are a few publications that have utilized the 3D scanning and 3D-printing technique to create various brain matrices for zebrafish, mice, and birds^14,15,16, those matrices are at a relatively small scale. There is no established MRI imaging series or detailed growth chart for the brains of neonatal piglets, especially for those 2 weeks and younger. Therefore, we created several brain slice templates using brains collected between day 5 and day 38, based on which an adjustable brain matrix system was established.

This protocol describes an innovative method for establishing neonatal piglet brain templates and developing acrylic plate-based, customizable brain matrix construction that can be used for coronal or sagittal sectioning of brains across a spectrum of sizes.

Protocol

In this article, the four piglet brains were obtained and prepared for histological staining after humane euthanasia following a protocol that has been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Kentucky for a separate study. All the procedures on animal subjects were approved, and appropriate care of animals was taken to minimize stress or pain associated with treatments or euthanasia.

1. Animals

1. Procure four naïve male neonatal (postnatal day 4, p4) piglets (body weight 1.5-1.8 kg) with a genetic background of 62.5% Landrace, 25% Large White, and 12.5% Yorkshire.
2. House the animals together in one dedicated room in a pigpen, with the support of a heat lamp and towel-covered floor. Feed them ad libitum with milk replacer for the first week and gradually transition to pellet food at 2-3 weeks.
3. Monitor the physiological activity and body weight and perform weekly MRI scans under isofluorane anesthesia.
4. Euthanize with pentobarbital overdose and transcardially perfuse with heparinized saline, followed by 10% neutral buffered formalin.
5. Dissect the brain out from the skull¹⁷, fix with formalin overnight, and then place in 30% sucrose in PBS for 3 days for cryopreservation.

2. Agarose gel molding

1. Submerge a glass container in a bucket of wet ice to its half-height (Figure 2A).
2. Use a heavy-duty plastic strip as a liner for the container and ensure the bottom and sidewalls are completely covered while leaving both ends at least 5 cm above the top edge.
3. Heat 150 mL of PBS in a glass beaker to 90 °C on a stirring hot plate with a magnet stirrer and gradually add and dissolve 6 g of agarose to make 4% agarose gel in PBS.
4. Pour a 0.5-1 cm thick layer into the glass container (Figure 2B) and wait until it solidifies.
5. Apply a thin layer of mineral oil topically on the brain (Figure 2C) and place the brain on the gel layer (Figure 2D).
6. Allow the agarose solution to cool to 50 °C. Embed the brain with cooled agarose gel (Figure 2E) and allow to solidify (Figure 2F), which generally takes ~10 min on wet ice.
7. Carefully remove the top layer of agarose gel above the brain (Figure 2G) with a surgical probe and extract the brain from the solidified mold (Figure 2H,I).
8. Heat 98 mL of PBS in another glass beaker to 90 °C on a stirring hot plate with a magnet stirrer, add 4 g of agarose and 2 mL of India Ink, and allow to stir and mix well. Pour 4% agarose with 2% India Ink into the cavity left by the extracted brain (Figure 2J) until full and allow it to solidify on wet ice for 15 min (Figure 2K).
9. Using the overhanging portions of the plastic strip, lift the plastic strip to remove the entire agarose block from the container (Figure 2L).

3. Agarose block slicing and template copying to scale paper

1. To protect the paper from moisture during the slicing procedure, place one piece of scale paper in between two 0.76 mm (0.03") thick transparent acrylic plates (Figure 3A), and place the laminated paper-acrylic plate set near the edge of the bench.
2. Secure corner squares to the bench with clamps in an appropriate position to keep two 5 mm thick acrylic plates standing and firmly holding the gel block in between with the desired orientation (sagittal in Figure 3). Ensure the edge of each vertical acrylic plate is aligned to the same line on the scale paper to use as a reference for slicing of the gel block. Use a weight to secure the gel block on the end opposite from where slicing will take place (Figure 3B).
3. Using the underlying scale paper as a guide, move the gel block forward from between the acrylic plates at the desired step (2 mm or 5 mm), hold the microtome blade with both hands, and slice the portion beyond the front edge of the acrylic plate (Figure 3B).
4. Collect each slice (Figure 3C), place it on a separate scale paper (Figure 3D), gently press the India ink dyed portion of the cast on the scale paper to transfer the outline (Figure 3E), trace the outline of the whole gel section using a pen, remove the gel section, and outline the India ink print also using a pen (Figure 3F). Pay attention to keeping the orientation of each slice and the copied template consistent.
5. Repeat steps 2.3 and 2.4 until all gel pieces are copied to the scale paper and numbered sequentially. Recheck the slices and their prints (representative images of coronal slices and prints in Figure 3G,H).

4. Acrylic plate carving

1. Cut grooves on the large acrylic plate with a Plexiglass cutting knife and break the large plates into 10 x 8.5 cm pieces.
   NOTE: The total number of pieces depends on the size of the brain, desired slice thickness, and slicing orientation. Generally, we prepare 25 pieces of 2 mm acrylic plates for either a coronal or sagittal matrix for neonatal piglet brains and 15 pieces of 5 mm plates for larger brains for easier handling.
2. Landmark three anchoring holes on the acrylic plate at x, y coordinates as measured from the left lower corner (in mm): (11, 42); (45, 15); and (88, 42).
3. Secure the acrylic plates with the support of corner squares and clamps on the platform of the drill press. Drill three anchoring holes at the aforementioned coordinates with an acrylic plate drill bit (Figure 4A). Draw a color line on the right side of all the plates to indicate orientation.
4. Remove the protective film from the acrylic plate and place it above the template on the scale paper, aligning the top edge of the acrylic plate to the top lines of the template that was copied to the scale paper. Trace the outline of the brain onto the acrylic plate with a permanent marker pen (Figure 4B). Label the plate sequentially as in the template.
5. Cut the acrylic plate on the band saw along the template lines (Figure 4C). Alternatively, use a rotary tool with a multi-purpose cutting kit and a wood carving tip to remove the unwanted areas.
6. Polish the cut edge using the rotary tool with a polishing sanding tip to smooth the edges left by the cutting tool (Figure 4D).
7. Recheck the cuts on the acrylic plates against the template on the scale paper (Figure 4E). Trim and polish if necessary.
8. Preassemble the matrix by stacking the cut plates in sequential order and check the alignment of the plates (Figure 4F).

5. Assembly and expansion of the brain matrix

1. Place three washers in between acrylic plates, each centered on one anchoring hole (Figure 5A). Glue them around the anchoring holes with superglue on the same side of all acrylic plates to reduce assembling time (Figure 5A).
2. Stack the acrylic plates sequentially, insert stainless bolts through each anchoring hole to assemble the matrix, and tighten the nuts to secure (Figure 5A).
3. Apply final polishing of the cut edges that will contact the brains to smooth across the plates.
4. Color code the top of the matrix to assist in identifying the steps during slicing. Alternately color the plate top edges with black, red, and then blank to form a pattern. Repeat this step until all plates are color-coded with the same pattern (Figure 5).
5. To adjust the geometry or change the cavity size to better fit certain brains, remove or add select acrylic plates (Figure 5B) to adapt to different brain sizes, which greatly increases the flexibility of these matrices and can save materials and effort.

6. Coronal or sagittal slicing of brains

1. Wrap one end of the high-profile cryostat blade with lab tape for at least 2 cm in length to protect the user's hand. Put the brain securely in the matrix cavity. Holding the wrapped end of the blade, carefully place the blade between the acrylic plates of the matrix, and then slice the brains into coronal or sagittal slices with the brain supported and secured by the other hand (Figure 6A,B).
   NOTE: Caution: 1) Since each of the brain parts has different textures after formalin fixation, caution should be exercised to apply even pressure on the blade. 2) Moreover, the piglet brain is large, so it is better to use a slicing motion to cut the brain than a direct "push-down" cutting motion. 3) It is critical to keep the brain snugly fitting in the matrix with the support of the other hand during the cutting to avoid any tilting or change in its orientation. Excessive pressure on the brain against the matrix should be avoided at all times.
2. Put all slices into a PBS-filled 6-well plate or stainless tray. Carefully lay the slices out in the same orientation (Figure 6C,D) and preserve or proceed as needed.

Results

One piglet was euthanized with pentobarbital overdose at the age of p5. The other three animals showed normal physiological activities and similar body weight gain (reaching 7.44-8.4 kg). Two piglets received weekly MRI scanning under isoflurane anesthesia until they were euthanized on p32. The fourth piglet remained naïve until euthanasia at p38.

The head size similarity between piglet and human fetus can be visualized in Figure 1A,B, which is at...

Discussion

This protocol presents the methodology for creating affordable and easy-to-build brain matrices that can be used to aid in the slicing of the piglet brain or any other species of large animals at the desired orientation and thickness. Importantly, as opposed to commercial "fixed-size" matrices that work with only a limited range of brain sizes, this approach allows the matrix to be conveniently adjusted or expanded to fit a wide range of brain sizes. In this report, a matrix built for a 30 g piglet brain was succ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1/4"-20 X 3-1/2" Stainless Phillips Truss Head Machine Screw, (25 pc), Coarse Thread, 18-8 (304) Stainless Steel, by Bolt Dropper	Bolt Dropper		https://www.amazon.com/ Stainless-Phillips-Machine -Bolt-Dropper/dp/B07D1L8 7VB/ref=sr_1_2?crid=38M PNERXG39Q3&keywords =1%2F4%22+3.5+inch+ stainless+bolts&qid=16427 82626&s=industrial&sprefix =1%2F4+3.5+inch+stainless +bolts%2Cindustrial%2C62 &sr=1-2
10 Pack of 8 x 10” PET Sheet/Plexiglass Panels 0.03” Thick	CALPALMY		https://www.amazon.com/dp /B084P4YSST/?coliid=I1S59 82XD9P6CB&colid=XJ78FAX D85BF&psc=1&ref_=lv_ov_lig_dp_it
3 Pack 3/16" (4.5mm) Clear Acrylic Sheet 8" x 12" Cast Plexiglass (0.177”) Thick Nominal Size AZM	AZM Displays		https://www.amazon.com/4-5mm -Acrylic-Plexiglass-Nominal-AZM /dp/B082CGYSCK/ref=sr_1_12? dchild=1&keywords=acrylic+sheet %2C+4.5mm&qid=1617572206&sr=8-12
3 Shoulder-Shortening Shim Flat Washer, 18-8 Stainless Steel, 1/2" Bolt Size, 0.501" ID, .750" OD, 0.016" Thick (Pack of 50)	Small Parts		https://www.amazon.com/Shoulder- Shortening-Washer-Stainless-Stee l-0-251/dp/B004K1FFZA/ref=sr_1_ 3?crid=1K8LO0VN4GLE&dchild=1 &keywords=Washers&pd_rd_r= 0ffe510e-abe5-4eae-b975-ed945 08ac71a&pd_rd_w=HuED5&pd_r d_wg=fixK7&pf_rd_p=b4950e17-f 2f6-494c-bba5-69a9d0aa3887&pf _rd_r=BRVGS2378CHD1WBC2E 4P&pid=3tntl8w&qid=1617573484 &refinements=p_n_feature_twenty _browse-bin%3A17420898011%2 Cp_n_feature_twenty-one_browse -bin%3A19047154011&s=industria l&sprefix=washer%2Bfor%2B%2C aps%2C202&sr=1-3&th=1
6 well plate	avantor	76520-650
8-32 Hex Nut, Coarse Thread Hexagon Nut, 18-8 Stainless Steel Hex Nut, Silver Tone,?100 Pcs?	Fullerkreg		https://www.amazon.com/Fullerkreg -Machine-Stainless-Bright-Quantity/ dp/B078S3J765/ref=sr_1_4?crid=4 VSSI5B9MJ2D&keywords=1%2F4 %22-20+Inch+Stainless+Steel+ Hex+nut+coarse+thread&qid=1 642782823&s=industrial&sprefix =1%2F4+-20+inch+stainless+ste el+hex+nut+coarse+thread%2Cin dustrial%2C61&sr=1-4
90 Degree Positioning Squares Aluminium Alloy	HINMAY		https://www.amazon.com/Positioni ng-Aluminium-Woodworking-Carp enter-Clamping/dp/B08294FZ7L/r ef=sr_1_6?crid=1I97TLVRYREPQ &dchild=1&keywords=corner+squ are&qid=1625683905&sprefix=co rner+squ%2Caps%2C182&sr=8-6
agarose	avantor	97062-244
band saw	SKIL
Birthright Milk Replacer (Pipestone)	Pipestone	SKU 8229	https://pipevet.com/birthright-baby-pig-milk-replacer
bucket	avantor	89233-340
Dremel 4300-5/40 High Performance Rotary Tool Kit	Dremel		https://www.amazon.com/dp/B0 1M1SJNVU/?coliid=IMLRI62ZL JTSJ&colid=2XZ304Y9M7PV7 &psc=1&ref_=lv_ov_lig_dp_it
Drill Bits for Plastic (Acrylic, plexiglass, ABS, lexan, Polycarbonate, PVC)	FTM		https://www.amazon.com/dp/B 07D1B3B51/?coliid=I26RS6UP UJ3P1Y&colid=2XZ304Y9M7P V7&psc=1&ref_=lv_ov_lig_dp_it
glass beaker x 2	avantor	470335-854
glass container	Wheaton staining dishes	WHEATON® 900303
Graph Paper Notebook: 1 mm thin and 10 mm thicker light gray grid lines (metric, 120 pages):			https://www.amazon.com/ Graph-Paper-Notebook- double-sided-non-perforated /dp/154319026X/ref=sr_1_13? dchild=1&keywords=millimeter +paper&qid=1617634163&sr=8-13
HACKER INSTRUMENTS MICROTOME KNIFE BLADE	HACKER INSTRUMENTS
heavy-duty plastic strip	avantor	89072-136
India Ink	speedball SUPER BLACK
IRWIN QUICK-GRIP Clamps, One-Handed, Mini Bar, 6-Inch, 4-Pack (1964758)	IRWIN		https://www.amazon.com/IRWIN -QUICK-GRIP-1964758-One- Handed-Clamp/dp/B001DSY4 QO/ref=sr_1_5?crid=3NJ320 86LJCS6&dchild=1&keywords =clamps+for+woodworking&qid =1617634545&sprefix=clamp% 2Caps%2C184&sr=8-5
Kreg KHC-Premium Face Clamp	Kreg		https://www.amazon.com/KREG -KHC-PREMIUM-Kreg-Face- Clamp/dp/B001DC9UR8/ref=sxin _11?ascsubtag=amzn1.osa.27 eddff2-544b-4c29-9242-220b3f 760cb0.ATVPDKIKX0DER.en_ US&creativeASIN=B001DC9UR 8&crid=3NJ32086LJCS6&cv_ct_ cx=clamps+for+woodworking&cv _ct_id=amzn1.osa.27eddff2-544b -4c29-9242-220b3f760cb0.ATVP DKIKX0DER.en_US&cv_ct_pg= search&cv_ct_we=asin&cv_ct_wn =osp-single-source-earns-comm& dchild=1&keywords=clamps+for+ woodworking&linkCode=oas&pd_r d_i=B001DC9UR8&pd_rd_r=eeaf7 178-eee3-4399-862a-479fffb6cfc3& pd_rd_w=W3Inj&pd_rd_wg=PKPUl& pf_rd_p=e666d5aa-04ca-46aa-86b0- 07ac28e037d4&pf_rd_r=22RKP4S4S SS5PYDM2ZFD&qid=1617634545&s prefix=clamp%2Caps%2C184&sr=1- 2-64f3a41a-73ca-403a-923c-8152c4 5485fe&tag=thisoldhouse05-20
lab tape	avantor	76276-016
Loctite 1363589 4-Gram Bottle Super Glue Ultra Gel Control Adhesive, 3-Pack	Loctite		https://www.amazon.com/Loctite- 1363589-4-Gram-Control-Adhesive/ dp/B01HPT0AWQ/ref=sr_1_13? keywords=superglue+power+glue +for+plastic&qid=1642782991&s prefix=superglue%2Caps%2C83&sr=8-13
Microtome High-Profile Disposable Blades	Optimal Scientific		https://www.amazon.com/Optimal -Scientific-Microtome-High-Profile -Disposable/dp/B08MCSN6SL/ref =sr_1_1?dchild=1&keywords=hig h+profile+cryostat+blade&qid=16 17592221&sr=8-1
mineral oil	Sigma-Aldrich	M8410-100ml
PBS	avantor	97062-818
polishing sanding tip	20 Pcs Carbide Burr Bits 1/8" Shank
Pre Starter Pellet Non-Med - #50 (Pipestone)	Pipestone	SKU 8228	https://pipevet.com/pre-starter-pellet-non-med-50-hubbard
Red Devil 1170 PLEXIGLASS Cutting TOOLCARDED	Red Devil		https://www.amazon.com/dp /B000BZZ1D0/?coliid=I3C0K3 AKD3TS75&colid=XJ78FAXD8 5BF&psc=1&ref_=lv_ov_lig_dp_it
Sharpie Permanent Markers | Chisel Tip Markers	Sharpie		https://www.amazon.com/ Sharpie-Permanent-Markers -Chisel-Classic/dp/B00006IFI A/ref=sr_1_8?crid=2GD78WJ5COF NN&keywords=permanent+marker s+assorted+colors&qid=16427831 06&sprefix=permanent+markers% 2Caps%2C129&sr=8-8
stirring hot plate	Corning	100493-718
stirring magent	VWR	58948-138
STYLEZONE 10 Pieces Transparent Clear Acrylic Sheets 8 x 10 Inch 0.08 Inch 2 mm-thick Acrylic Board	Stylezone		https://www.amazon.com/dp/ B08GJYS4TT/?coliid=I11RLE V42BKEFB&colid=XJ78FAX D85BF&psc=1&ref_=lv_ov_lig_dp_it
surgial tray	avantor	10193-194
wet ice	lab made
wood carvign tip	Wood Carving Tools for Rotary Tool		https://www.amazon.com/ stores/page/28E50A85-D8C 1-4669-8272-D5D435EA126F