Name: Fabrication of an Expandable Brain Matrix Customizable Across Developmental Stages (Video) | JoVE Journal
Uploaded: 2026-02-20T14:40:00.000+00:00
Duration: 11 min 35 s
Description: Learn to fabricate an expandable brain matrix for neonatal piglet brain slicing using acrylic plates and agarose gel molding. This cost-effective method accommodates developmental growth across multiple species.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by the National Institutes of Health (NIH) #R21-NS114771 (K.E.S.),&#160;#R56-NS117587, #R01-EB028792, #R01-HD101508, #R21-HD091118, #R41-NS122722, #R42-MH135825 (G. Y.), American Heart Association (AHA) #16GRNT30820006 (G.Y.) and #14SDG20480186 (L.C.), and the University of Kentucky Neuroscience Research Priority Area (L.C.). The na&#239;ve male neonatal pigs were purchased through the Division of Laboratory Animal Resources (DLAR) from the Swine Center of the College of Agriculture, University of Kentucky.

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
<ol>
	<li>Procure four na&#239;ve 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.</li>
	<li>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.</li>
	<li>Monitor the physiological activity and body weight and perform weekly MRI scans under isofluorane anesthesia.</li>
	<li>Euthanize with pentobarbital overdose and transcardially perfuse with heparinized saline, followed by 10% neutral buffered formalin.</li>
	<li>Dissect the brain out from the skull17, fix with formalin overnight, and then place in 30% sucrose in PBS for 3 days for cryopreservation.</li>
</ol>
2. Agarose gel molding
<ol>
	<li>Submerge a&#160;glass container in a bucket of wet ice to its half-height (Figure 2A).</li>
	<li>Use a&#160;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.</li>
	<li>Heat 150 mL of PBS in a glass beaker to 90 &#176;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.</li>
	<li>Pour a 0.5-1 cm thick layer into the glass container (Figure 2B) and wait until it solidifies.</li>
	<li>Apply a thin layer of mineral oil topically on the brain (Figure 2C) and place the brain on the gel layer (Figure 2D).</li>
	<li>Allow the agarose solution to cool to 50 &#176;C. Embed the brain with cooled agarose gel (Figure 2E) and allow to solidify (Figure 2F), which generally takes ~10 min on wet ice.</li>
	<li>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).</li>
	<li>Heat 98 mL of PBS in another glass beaker to 90 &#176;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).</li>
	<li>Using the overhanging portions of the plastic strip, lift the plastic strip to remove the entire agarose block from the container (Figure 2L).</li>
</ol>
3. Agarose block slicing and template copying to scale paper
<ol>
	<li>To protect the paper from moisture during the slicing procedure, place one piece of scale paper in between two 0.76 mm (0.03&#34;) thick transparent acrylic plates (Figure 3A), and place the laminated paper-acrylic plate set near the edge of the bench.</li>
	<li>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&#160;vertical acrylic plate&#160;is&#160;aligned to the same line on the scale paper to use as a reference for slicing of the gel block. Use a&#160;weight to secure the gel block on the end opposite from where slicing will take place (Figure 3B).</li>
	<li>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).</li>
	<li>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.</li>
	<li>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).</li>
</ol>
4. Acrylic plate carving
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Polish the cut edge using the rotary tool with a polishing sanding tip to smooth the edges left by the cutting tool (Figure 4D).</li>
	<li>Recheck the cuts on the acrylic plates against the template on the scale paper (Figure 4E). Trim and polish if necessary.</li>
	<li>Preassemble the matrix by stacking the cut plates in sequential order and check the alignment of the plates (Figure 4F).</li>
</ol>
5. Assembly and expansion of the brain matrix
<ol>
	<li>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).</li>
	<li>Stack the acrylic plates sequentially, insert stainless bolts through each anchoring hole to assemble the matrix, and tighten the nuts to secure (Figure 5A).</li>
	<li>Apply final polishing of the cut edges that will contact the brains to smooth across the plates.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
6. Coronal or sagittal slicing of brains
<ol>
	<li>Wrap one end of the high-profile cryostat blade with lab tape for at least 2 cm in length to protect the user&#39;s hand. Put the brain securely in the matrix cavity. Holding the wrapped end of the blade, carefully place&#160;the blade between the acrylic plates of the matrix, and then slice&#160;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 &#34;push-down&#34; 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.</li>
	<li>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.</li>
</ol>

<ol><li>Jelsing, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+postnatal+development+of+neocortical+neurons+and+glial+cells+in+the+Gottingen+minipig+and+the+domestic+pig+brain%5BTitle%5D)+AND+%22J+Exp+Biol%22%5BJournal%5D)">The postnatal development of neocortical neurons and glial cells in the Gottingen minipig and the domestic pig brain.</a> J Exp Biol. 209 (Pt 8), 1454-1462 (2006).</li>
<li>Conrad, M. S., Dilger, R. N., Johnson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+growth+of+the+domestic+pig+%28Sus+scrofa%29+from+2+to+24+weeks+of+age%3A+a+longitudinal+MRI+study%5BTitle%5D)+AND+%22Dev+Neurosci%22%5BJournal%5D)">Brain growth of the domestic pig (Sus scrofa) from 2 to 24 weeks of age: a longitudinal MRI study.</a> Dev Neurosci. 34 (4), 291-298 (2012).</li>
<li>Conrad, M. S., Dilger, R. N., Nickolls, A., Johnson, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magnetic+resonance+imaging+of+the+neonatal+piglet+brain%5BTitle%5D)+AND+%22Pediatr+Res%22%5BJournal%5D)">Magnetic resonance imaging of the neonatal piglet brain.</a> Pediatr Res. 71 (2), 179-184 (2012).</li>
<li>Dobbing, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+later+growth+of+the+brain+and+its+vulnerability%5BTitle%5D)+AND+%22Pediatrics%22%5BJournal%5D)">The later growth of the brain and its vulnerability.</a> Pediatrics. 53 (1), 2-6 (1974).</li>
<li>Sorby-Adams, A. J., Vink, R., Turner, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large+animal+models+of+stroke+and+traumatic+brain+injury+as+translational+tools%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">Large animal models of stroke and traumatic brain injury as translational tools.</a> Am J Physiol Regul Integr Comp Physiol. 315 (2), R165-R190 (2018).</li>
<li>Sauleau, P., Lapouble, E., Val-Laillet, D., Malbert, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+pig+model+in+brain+imaging+and+neurosurgery%5BTitle%5D)+AND+%22Animal%22%5BJournal%5D)">The pig model in brain imaging and neurosurgery.</a> Animal. 3 (8), 1138-1151 (2009).</li>
<li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Speckle+contrast+diffuse+correlation+tomography+of+cerebral+blood+flow+in+perinatal+disease+model+of+neonatal+piglets%5BTitle%5D)+AND+%22J+Biophotonics%22%5BJournal%5D)">Speckle contrast diffuse correlation tomography of cerebral blood flow in perinatal disease model of neonatal piglets.</a> J Biophotonics. 14 (4), 2021(2021).</li>
<li>Dobbing, J., Sands, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+growth+and+development+of+human+brain%5BTitle%5D)+AND+%22Arch+Dis+Child%22%5BJournal%5D)">Quantitative growth and development of human brain.</a> Arch Dis Child. 48 (10), 757-767 (1973).</li>
<li>Lossi, L., D'Angelo, L., De Girolamo, P., Merighi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomical+features+for+an+adequate+choice+of+experimental+animal+model+in+biomedicine%3A+II.+small+laboratory+rodents%2C+rabbit%2C+and+pig%5BTitle%5D)+AND+%22Ann+Anat%22%5BJournal%5D)">Anatomical features for an adequate choice of experimental animal model in biomedicine: II. small laboratory rodents, rabbit, and pig.</a> Ann Anat. 204, 11-28 (2016).</li>
<li>Lind, N. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+pigs+in+neuroscience%3A+modeling+brain+disorders%5BTitle%5D)+AND+%22Neurosci+Biobehav+Rev%22%5BJournal%5D)">The use of pigs in neuroscience: modeling brain disorders.</a> Neurosci Biobehav Rev. 31 (5), 728-751 (2007).</li>
<li>Andersen, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Delayed+growth%2C+motor+function+and+learning+in+preterm+pigs+during+early+postnatal+life%5BTitle%5D)+AND+%22Am+J+Physiol+Regul+Integr+Comp+Physiol%22%5BJournal%5D)">Delayed growth, motor function and learning in preterm pigs during early postnatal life.</a> Am J Physiol Regul Integr Comp Physiol. 310 (6), R481-R492 (2016).</li>
<li>Desantis, S., Minervini, S., Zallocco, L., Cozzi, B., Pirone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+changes+in+the+primary+motor+cortex+of+newborn+to+adult+domestic+pig+Sus+scrofa+domesticus%5BTitle%5D)+AND+%22Animals+%28Basel%29%22%5BJournal%5D)">Age-related changes in the primary motor cortex of newborn to adult domestic pig Sus scrofa domesticus.</a> Animals (Basel). 11 (7), 2019(2021).</li>
<li>Pond, W. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perinatal+ontogeny+of+brain+growth+in+the+domestic+pig%5BTitle%5D)+AND+%22Proc+Soc+Exp+Biol+Med%22%5BJournal%5D)">Perinatal ontogeny of brain growth in the domestic pig.</a> Proc Soc Exp Biol Med. 223 (1), 102-108 (2000).</li>
<li>Huang, C. C., Chen, Y. Y., Fang, Y. T., Chen, Y. C., Hung, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generating+brain+matrices+for+zebra+finch+brain+sectioning+using+three-dimensional+printing+technology%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">Generating brain matrices for zebra finch brain sectioning using three-dimensional printing technology.</a> J Neurosci Methods. 327, 108399(2019).</li>
<li>Yamazaki, Y., Yuguchi, M., Honjo, B., Isokawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+and+fabrication+of+customized+brain+slice+matrices+using+CAD+and+3D+printing+technology%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Design and fabrication of customized brain slice matrices using CAD and 3D printing technology.</a> PLoS One. 20 (1), e0317616(2025).</li>
<li>Lattin, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+3D-printed+modular+device+for+imaging+the+brain+of+small+birds%5BTitle%5D)+AND+%22J+Neurosci+Methods%22%5BJournal%5D)">A 3D-printed modular device for imaging the brain of small birds.</a> J Neurosci Methods. 293, 183-190 (2018).</li>
<li>Fleming, S. A., Monaikul, S., Mudd, A. T., Jacob, R., Dilger, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extraction+and+dissection+of+the+domesticated+pig+brain%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Extraction and dissection of the domesticated pig brain.</a> J Vis Exp. (170), e62030(2021).</li>
</ol>

The authors have no conflicts of interest to disclose.

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 &#34;fixed-size&#34; 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...

Piglet brain development closely mimics that of humans1,2,3,4. 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 birds14,15,16, 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.

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

fabrication of an expandable brain matrix customizable across developmental stages

Fabrication of an Expandable Brain Matrix Customizable Across Developmental Stages

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.

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...

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.

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 ...

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.

fabrication-an-expandable-brain-matrix-customizable-across

Research

JoVE Journal

Neuroscience

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates.  These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used  to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered "living scaffolds", exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Described below is a method for implantation of multiple polymer electrode arrays across anatomically distant brain regions for chronic electrophysiological recording in freely moving rats. Preparation and surgical implantation are described in detail, with emphasis on design principles to guide adaptation of these methods for use in other species.

Chronic Implantation of Multiple Flexible Polymer Electrode Arrays

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

The method outlined below aims to provide a comprehensive protocol for the preparation of nonhuman primate (NHP) neurosurgery using a novel combination of three-dimensional (3D) printing methods and MRI data extraction.

A MRI-Based Toolbox for Neurosurgical Planning in Nonhuman Primates

This work presents a bottom-up approach to the engineering of local magnetic forces for control of neuronal organization. Neuron-like cells loaded with magnetic nanoparticles (MNPs) are plated atop and controlled by a micro-patterned platform with perpendicular magnetization. Also described are magnetic characterization, MNP cellular uptake, cell viability, and statistical analysis.

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons

This work describes a protocol for the freeform embedded 3D printing of neural stem cells inside self-healing annealable particle-extracellular matrix composites. The protocol enables the programmable patterning of interconnected human neural tissue constructs with high fidelity.

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

This paper outlines automated processes for nonhuman primate neurosurgical planning based on magnetic resonance imaging (MRI) scans. These techniques use procedural steps in programming and design platforms to support customized implant design for NHPs. The validity of each component can then be confirmed using three-dimensional (3D) printed life-size anatomical models.

A Neural Implant Design Toolbox for Nonhuman Primates

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Learn to fabricate an expandable brain matrix for neonatal piglet brain slicing using acrylic plates and agarose gel molding. This cost-effective method accommodates developmental growth across multiple species.

Fabrication of an Expandable Brain Matrix Customizable Across Developmental Stages

Explore More Videos