The authors would like to thank the Morris Animal Foundation for their funding of this project.

Equine enteric glial primary cultures were obtained from three horses humanely euthanized with an overdose of a barbiturate for reasons unrelated to this study. The horses selected for the culture studies were adult horses with no current history of gastrointestinal disease.
1. Submucosal equine enteric glial primary culture
<ol>
	<li>Grow equine epithelial monolayers from jejunal crypts obtained from three separate adult equine horses.
	<ol>
		<li>Coat 24-well plates with coating solution for 2 h at 37 &#176;C or overnight at room temperature, wash them 2x with sterile water, and store them at room temperature until the cells are ready for plating.</li>
		<li>Following humane euthanasia, within 60 min, remove a section of the jejunum and place it in cold Ringer&#39;s solution on ice.</li>
		<li>Remove a 10 cm portion from the jejunum and open it at the antimesenteric border to keep non-lymphoid regions at the center of the tissue. Trim the portion to an approximately 7 cm x 7 cm section. Rinse the tissue well in fresh Ringer&#39;s solution prior to dissection.</li>
		<li>Pin the tissue on a silicone elastomer-coated Petri dish in cold Ringer&#39;s solution or PBS mucosal side up, stretching the tissue as much as possible (Figure 1A).</li>
		<li>Remove the intestinal villi with curved forceps (Figure 1B) followed by the lamina propria layer from the approximately 5 cm x 5 cm non-lymphoid region (Figure 1C). 
		NOTE: These layers separate easily from the submucosa (Figure 1B).</li>
		<li>Remove the submucosa in one sheet by freeing it with microdissection scissors at one corner and observe the natural separation while peeling the submucosa away from the inner circular muscle layer of the jejunum (Figure 1D).</li>
		<li>Mince the collected submucosal layer into 2-5 mm pieces and place them into a 50 mL conical tube containing 5 mL of &#34;Organo-FBS&#34; (Table 1), 0.825 mg of collagenase, 5 mg of protease, and 20 mg of bovine serum albumin. Incubate the tissue for 2-3 h at 37 &#176;C for enzymatic digestion.</li>
		<li>Following incubation, add 10 mL of room temperature &#34;Organo + FBS&#34; to stop the enzyme action. Pipette the tissue mixture up and down approximately 15x with a 10 mL serological pipette to dissociate cells from the tissue and then centrifuge at 3,000 &#215; g for 3 min at room temperature.</li>
		<li>Discard the supernatant and resuspend the pellet with 10 mL room temperature PBS by pipetting up and down again approximately 15x with a 10 mL serological pipette to dissociate cells from the tissue.</li>
		<li>Centrifuge the conical tube at 3,000 &#215; g for 3 min at room temperature. Discard the supernatant and resuspend the pellet with 10 mL of room temperature &#34;Organo + FBS&#34; by pipetting up and down approximately 15x with a 10 mL serological pipette to dissociate cells from the tissue.</li>
		<li>Filter the cells serially through a 100 &#181;m, a 70 &#181;m, and then a 40 &#181;m pore-size cell strainer.</li>
		<li>Centrifuge the filtered cells and media at 3,000 &#215; g for 3 min at room temperature. Discard the supernatant and resuspend the pellet in 1 mL of &#34;Organo + FBS.&#34;</li>
		<li>Stain with trypan blue to identify live cells and count them with a hemocytometer.</li>
		<li>Seed approximately 400,000 cells in 300 &#181;L of &#34;Organo + FBS&#34; in each well of a 24-well plate and add growth factors to each well: N2 5 &#181;L/well, G5 5 &#181;L/well, and B27 10 &#181;L/well. Place the 24-well plate in a 37 &#176;C incubator with 5% CO2 to allow the cells to adhere. Twenty-four hours later, change the media to &#34;glia media&#34; (Table 1) with the above growth factors and change every 48 h (Figure 2).</li>
	</ol>
	</li>
</ol>
2. Immunofluorescent cytology
<ol>
	<li>Grow the enteric glial cells from the primary culture (P0) (step 2.1.14) to 70-80% confluence and passage them once (P1).</li>
	<li>Fix the primary culture (P0) cells in the wells using 4% paraformaldehyde for 5 min prior to the application of 0.1% PBS azide.</li>
	<li>Prepare saturation solution consisting of PBS azide, 4% donkey serum, and 0.5% Triton-X. Add 300 &#181;L of saturation solution to the wells to permeabilize the cells for 2 h at room temperature.</li>
	<li>Add the primary antibody for the glial marker, glial fibrillary acidic protein (GFAP), and the primary antibody for the fibroblast marker, alpha smooth muscle actin, at 1:1,000 dilutions in saturation solution for overnight incubation at 4 oC.</li>
	<li>Wash 3x with PBS prior to adding secondary antibody for donkey anti-rabbit IgG Alexa Fluor 594 and goat anti-mouse IgG Alexa Flour 488 at a 1:500 dilution in saturation solution for 2-3 h.</li>
	<li>Stain the nuclei with 4&#39;,6-diamidino-2-phenylindole (DAPI) at a 1:1,000 dilution in PBS for 5 min.</li>
	<li>Following three washes with PBS, store the cells in 0.1% PBS azide.</li>
</ol>
3. Submucosal equine enteric glial primary culture IL-1&#946; exposure
<ol>
	<li>When cells from the first passage (P1) reach 70-80% confluence, perform the IL-1&#946; exposure experiment. Expose the wells to 0 ng, 10 ng, and 25 ng of IL-1&#946; for 24 h. Following 24 h, bank and store the media from each well at -80 &#176;C.</li>
</ol>
4. Equine organoid culture for production of enterocyte monolayers
NOTE: Following the Stewart et al. protocol published in 2018, banked equine intestinal crypts and buds that had been cryobanked were expanded for 2D enteroid studies10.
<ol>
	<li>Use approximately 700-1,000 buds/crypt fragments (40,000-50,000 cells when dissociated into single cells) per transwell insert. Coat the transwell inserts with 42 &#181;L of 0.05% Matrigel prior to the addition of the enterocytes. Allow the Matrigel to be fixed on the insert by incubating for 1 h at 37 oC. Aspirate the extra medium and allow the transwells to dry for 30 min uncovered. Add 200 &#181;L of DMEM-F12 medium to each transwell insert and store the plate at 37 &#176;C until use.</li>
	<li>Wash the Matrigel patties containing the organoids with PBS and then expose them to 500 &#181;L of Cell Recovery while on ice. Pipette them repeatedly to ensure full collection of cells into a 15 mL tube.</li>
	<li>Centrifuge the tube at 300 &#215; g for 5 min at 4 &#176;C., remove the supernatant, and suspend the cell pellet in the target volume of intestinal epithelial stem cell (IESC) medium. Calculate the target volume by multiplying the desired number of monolayers by 200 &#181;L of medium. (Table 1).</li>
	<li>Precoat a 1 mL syringe in IESC media and aspirate the cell suspension through a 16 G needle. Replace the 16 G needle with a 28 G needle and eject the cell suspension through it to encourage separation of the organoids.</li>
	<li>Place 200 &#181;L of the resulting cell suspension into the apical side of the coated transwells and 500 &#181;L of IESC as well as previously described growth factors on the basal side of the transwell insert10. Change the media and growth factors every 24-48 h until confluence is reached. Define confluence as the cell density corresponding to a TEER of 1,000 ohmsxcm2,11. For measurement, see step 5.1.</li>
</ol>
5. Equine enterocyte monolayer permeability following exposure to inflammatory cytokines and equine enteric glial products
<ol>
	<li>Use a dual-electrode TEER-measuring chamber to quantify the TEER across the monolayers and establish a base reading across each transwell permeable membrane. Ensure that the IESC media and transwell have 15 min to come to room temperature prior to taking measurements. Fill the culture cup with 1.5 mL of IESC media and ensure exactly 200 &#181;L of media is present within the transwell insert so that the fluid lines match at the time of measurement.</li>
	<li>Expose the basolateral aspect of the transwell inserts to either inflammatory cytokines of 25 ng IL-1&#946;, 10 ng and 25 ng interleukin 6 (IL-6) per well, or glial products of cultures exposed to 10 ng or 25 ng IL-1&#946;. Expose the basolateral aspect of the control transwell inserts to glia media, and glia media from cultures not exposed to IL-1&#946;.</li>
	<li>Measure the TEER for 45 min at 10-15 min intervals by placing each transwell into the culture cup.</li>
</ol>
6. Statistics
<ol>
	<li>Calculate the fold change of the TEER of each transwell for the time points: 0, 10, 20, 30, and 45 min.</li>
	<li>Utilizing the software of choice (see Table of Materials), compare the correlations between decreased TEER and basolateral exposure to IL-1&#946; (25 ng), IL-6 (10 ng, 25 ng, and 100 ng), and glia media (exposed to 0 ng IL-1&#946;, 10 ng IL-1&#946;, and 25 ng IL-1&#946;) to control media using an unpaired one-tailed t-test.</li>
</ol>

Studying Enteric Glial-Epithelial Interactions in Colic-Related Inflammation

<ol>
	<li>Tinker, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+study+of+equine+colic+incidence+and+mortality.">Prospective study of equine colic incidence and mortality.</a> Equine Vet J. 29 (6), 448-453 (1997).</li><li>Morton, A. J., Blikslager, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+and+postoperative+factors+influencing+short-term+survival+of+horses+following+small+intestinal+resection:+92+cases+(1994-2001).">Surgical and postoperative factors influencing short-term survival of horses following small intestinal resection: 92 cases (1994-2001).</a> Equine Vet J. 34 (5), 450-454 (2002).</li><li>French, N. P., Smith, J., Edwards, G. B., Proudman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equine+surgical+colic:+risk+factors+for+postoperative+complications.">Equine surgical colic: risk factors for postoperative complications.</a> Equine Vet J. 34 (5), 444-449 (2002).</li><li>Lisowski, Z. M., Lefevre, L., Mair, T. S., Clark, E. L., Hudson, N. P. H., Hume, D. A., Pirie, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+quantitative+real-time+PCR+to+determine+the+local+inflammatory+response+in+the+intestinal+mucosa+and+muscularis+of+horses+undergoing+small+intestinal+resection.">Use of quantitative real-time PCR to determine the local inflammatory response in the intestinal mucosa and muscularis of horses undergoing small intestinal resection.</a> Equine Vet J. 54 (1), 52-62 (2022).</li><li>Sheats, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+equine+SIRS,+sepsis,+and+neutrophils.">A comparative review of equine SIRS, sepsis, and neutrophils.</a> Front Vet Sci. 6, 69 (2019).</li><li>Mazzotta, E., Villalobos-Hernandez, E. C., Fiorda-Diaz, J., Harzman, A., Christofi, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+ileus+and+postoperative+gastrointestinal+tract+dysfunction:+pathogenic+mechanisms+and+novel+treatment+strategies+beyond+colorectal+enhanced+recovery+after+surgery+protocols.">Postoperative ileus and postoperative gastrointestinal tract dysfunction: pathogenic mechanisms and novel treatment strategies beyond colorectal enhanced recovery after surgery protocols.</a> Front Pharm. 11, 583422 (2020).</li><li>Stoffels, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postoperative+ileus+involves+interleukin-1+receptor+signaling+in+enteric+glia.">Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.</a> Gastroenterology. 146 (1), 176-187 (2014).</li><li>Yu, Y. B., Li, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glial+cells+and+their+role+in+the+intestinal+epithelial+barrier.">Enteric glial cells and their role in the intestinal epithelial barrier.</a> World J Gastroenterol. 20 (32), 11273-11280 (2014).</li><li>Savidge, T. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+regulate+intestinal+barrier+function+and+inflammation+via+release+of+S-nitrosoglutathione.">Enteric glia regulate intestinal barrier function and inflammation via release of S-nitrosoglutathione.</a> Gastroenterology. 132 (4), 1344-1358 (2007).</li><li>Stewart, A. S., Freund, J. M., Gonzalez, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+three-dimensional+culture+of+equine+intestinal+epithelial+stem+cells.">Advanced three-dimensional culture of equine intestinal epithelial stem cells.</a> Equine Vet J. 50 (2), 241-248 (2018).</li><li>Hellman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+equine+enteroids+and+enteroid-derived+2D+monolayers+that+are+responsive+to+microbial+mimics.">Generation of equine enteroids and enteroid-derived 2D monolayers that are responsive to microbial mimics.</a> Vet Res. 52 (1), 108 (2021).</li><li>Al-Sadi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-6+modulation+of+intestinal+epithelial+tight+junction+permeability+is+mediated+by+JNK+pathway+activation+of+claudin-2+gene.">Interleukin-6 modulation of intestinal epithelial tight junction permeability is mediated by JNK pathway activation of claudin-2 gene.</a> PLoS One. 9 (3), e85345 (2014).</li><li>Ochoa-Cortes, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glial+cells:+A+new+frontier+in+neurogastroenterology+and+clinical+target+for+inflammatory+bowel+diseases.">Enteric glial cells: A new frontier in neurogastroenterology and clinical target for inflammatory bowel diseases.</a> Inflamm Bowel Dis. 22 (2), 433-449 (2016).</li><li>Ziegler, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glial+cell+network+function+is+required+for+epithelial+barrier+restitution+following+intestinal+ischemic+injury+in+the+early+postnatal+period.">Enteric glial cell network function is required for epithelial barrier restitution following intestinal ischemic injury in the early postnatal period.</a> Am J Physiol Gastrointest Liver Physiol. 326 (3), G228-G246 (2024).</li><li>Grubi&#353;i&#263;, V., Gulbransen, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia:+the+most+alimentary+of+all+glia.">Enteric glia: the most alimentary of all glia.</a> J Physiol. 595 (2), 557-570 (2017).</li></ol>

The authors have no conflicts of interest to disclose.

The aim of this study was to develop a repeatable method of primary culture of equine submucosal enteric glia and demonstrate its application to model epithelial-glial interactions at the time of colic. Enteric glia isolation and culture, which is novel in the horse, has proven beneficial in understanding intestinal disease pathways in pig and rodent models and in humans6,7,13,14. Studying this...

Equine colic is the most prevalent medical presenting complaint for emergency consultation1. With up to 17% of those horses requiring surgical correction, efforts to increase postoperative outcomes should be at the forefront of equine medical research2. Currently, postoperative colic patients experience a high risk of several life-threatening disorders including sepsis/endotoxemic shock (12.3% of patients) and postoperative ileus (13.7% of patients)3. Despite progress in treatment for postoperative complications, there continues to be a need for advanced treatments for preventing or treating these conditions.
Recent research has highlighted the local and systemic inflammatory status of colic patients4. For example, proinflammatory proteins such as tumor necrosis factor (TNF) alpha, interleukin 1&#946; (IL-1&#946;), interleukin 6 (IL-6), and monocyte chemoattractant protein-1, have all been shown to be significantly increased in expression in colic intestinal mucosa versus normal intestinal tissue4. From a systemic point of view, it has been demonstrated that increased TNF alpha in the intestinal tissue is correlated with an increased risk of postoperative nasogastric reflux greater than 2 L, a general measurement of postoperative dysmotility4. It is also known that the administration of interleukin IL-1&#946; and TNF alpha is capable of inducing clinical signs of septic shock5.
A potential explanation for the inflammatory status of colic intestinal tissue and its link to postoperative complications is the intestinal epithelial barrier. In health, the tight junction complexes linking the single layer of columnar epithelium that lines the intestinal tract provide a functional barrier to limit the luminal content and its bacterial components from reaching the submucosal space and bloodstream. However, the distension and damage caused by colic-associated intestinal obstruction and intestinal manipulation during surgery may disrupt this intestinal barrier function.
In terms of the functional components of the intestinal wall as a whole, the submucosal enteric glial network within the enteric nervous system has been shown to be crucial in the pathophysiological development of postoperative complications associated with inflammatory pathways and may provide a specific therapeutic target6,7,8,9. Not only are enteric glia present throughout the entire gastrointestinal tract, but they act as sensors of the intestinal environment, influence signaling with numerous cell types of the intestinal wall, and directly regulate the intestinal barrier6. It is, therefore, justifiable to presume that these potent sensors of the intestine would be activated by injury and inflammation and could produce an acute response such as alterations in barrier permeability.
This study is the first to describe the culture of equine enteric glia and, more specifically, the role of inflammatory equine enteric glia on intestinal barrier function. Here, we present methods of primary culture of equine submucosal enteric glia and their response to exposure to inflammatory IL-1&#946;, evaluation of the effect of enteric glial products post IL-1&#946; exposure on the permeability of equine enterocyte monolayers, and possible blockade through the application of equine serum.

<table><tbody><tr><td>1 M HEPES buffer</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>10 mM HEPES</td><td>Life Technologies</td><td>15630-106</td><td></td></tr><tr><td>2 mM GlutaMAX</td><td>Life Technologies</td><td>25050-061</td><td></td></tr><tr><td>4&amp;rsquo;6-Diaminidino-2-Phenylindol</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>Advanced DMEM/F12</td><td>Life Technologies</td><td>12634-010</td><td></td></tr><tr><td>Alpha smooth muscle actin antibody</td><td>Abcam</td><td>7817</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>AA9529</td><td>4.4 g/mL stock aliquots, final concentration 1.1 &amp;micro;g/mL</td></tr><tr><td>Anti-Antimicotic 1x</td><td>Gibco</td><td>15240-096</td><td></td></tr><tr><td>B27</td><td>Gibco</td><td>12587010</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>AA3311</td><td></td></tr><tr><td>BSA 50 mg/mL stock solution</td><td>Sigma</td><td>A3311</td><td></td></tr><tr><td>CaCL2</td><td>ACROS Organiics</td><td>206791000</td><td>Component of Equine Ringer &amp;lsquo;s Stock 1: combine with other ingredients, then add 100 mL of this stock to a graduated cylinder and dilute to 1L with deionized water. Adjust pH to 7.4 with 5% CO2.&lt;br/&gt; Combine with Equine Ringer&amp;rsquo;s Stock 2 to make complete &amp;ldquo;Ringer&amp;rsquo;s Solution&amp;rdquo;.</td></tr><tr><td>Collagenase</td><td>Sigma</td><td>9891</td><td></td></tr><tr><td>DMEM-F12 media</td><td>Thermo Fisher</td><td>11320033</td><td></td></tr><tr><td>Donkey anti-rabbit IgG Alexa Fluor 594</td><td>Invitrogen</td><td>21207</td><td></td></tr><tr><td>EVOM EndOhm dual electrode TEER-measuring chamber</td><td>World Precision Instruments</td><td>EVM-EL-03-01</td><td></td></tr><tr><td>EVOM Manual for TEER Measurement</td><td>World Precision Instruments</td><td>EVM-MT-03-01</td><td></td></tr><tr><td>G5</td><td>Gibco</td><td>17503012</td><td></td></tr><tr><td>Gentamicin solution</td><td>Sigma</td><td>G1272</td><td>Final concentration 20 &amp;micro;g/mL</td></tr><tr><td>GFAP antibody</td><td>Abcam</td><td>4674</td><td></td></tr><tr><td>Goat anti-mouse IgG Alexa Fluor 488</td><td>Invitrogen</td><td>28175</td><td></td></tr><tr><td>IL-1&amp;beta; ELISA</td><td>Thermo Fisher</td><td>ESIL1B</td><td></td></tr><tr><td>KCl</td><td>Thermo Fisher</td><td>P330-500</td><td>Component of Equine Ringer&amp;rsquo;s Stock 1: combine with other ingredients, then add 100 mL of this stock to a graduated cylinder and dilute to 1L with deionized water. Adjust pH to 7.4 with 5% CO2.&lt;br/&gt; Combine with Equine Ringer&amp;rsquo;s Stock 2 to make complete &amp;ldquo;Ringer&amp;rsquo;s Solution&amp;rdquo;.</td></tr><tr><td>L-glutamine solution</td><td>Corning</td><td>25-00-Cl</td><td></td></tr><tr><td>Matrigel</td><td>BD Bioscience</td><td>354277</td><td></td></tr><tr><td>MgCl2</td><td>Thermo Fisher</td><td>M33-500</td><td>Component of Equine Ringer&amp;rsquo;s Stock 1: combine with other ingredients, then add 100 mL of this stock to a graduated cylinder and dilute to 1L with deionized water. Adjust pH to 7.4 with 5% CO2.&lt;br/&gt; Combine with Equine Ringer&amp;rsquo;s Stock 2 to make complete &amp;ldquo;Ringer&amp;rsquo;s Solution&amp;rdquo;.</td></tr><tr><td>N2</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>Na2HPO4</td><td>Thermo Fisher</td><td>BP332-1</td><td>Component of Equine Ringer&amp;rsquo;s Stock 2: combine with other ingredients, then add 100 mL of this stock to a graduated cylinder and dilute to 1L with deionized water. Adjust pH to 7.4 with 5% CO2.&lt;br/&gt; Combine with Equine Ringer&amp;rsquo;s Stock 1 to make complete &amp;ldquo;Ringer&amp;rsquo;s Solution&amp;rdquo;.</td></tr><tr><td>NaCl</td><td>Thermo Fisher</td><td>S271-10</td><td>Component of Equine Ringer&amp;rsquo;s Stock 1: combine with other ingredients, then add 100 mL of this stock to a graduated cylinder and dilute to 1L with deionized water. Adjust pH to 7.4 with 5% CO2.&lt;br/&gt; Combine with Equine Ringer&amp;rsquo;s Stock 2 to make complete &amp;ldquo;Ringer&amp;rsquo;s Solution&amp;rdquo;.</td></tr><tr><td>NaH2PO4</td><td>Thermo Fisher</td><td>BP329-500</td><td>Component of Equine Ringer&amp;rsquo;s Stock 2: combine with other ingredients, then add 100 mL of this stock to a graduated cylinder and dilute to 1L with deionized water. Adjust pH to 7.4 with 5% CO2.&lt;br/&gt; Combine with Equine Ringer&amp;rsquo;s Stock 1 to make complete &amp;ldquo;Ringer&amp;rsquo;s Solution&amp;rdquo;.</td></tr><tr><td>NaHCO3</td><td>Thermo Fisher</td><td>S637-212</td><td>Component of Equine Ringer&amp;rsquo;s Stock 2: combine with other ingredients, then add 100 mL of this stock to a graduated cylinder and dilute to 1L with deionized water. Adjust pH to 7.4 with 5% CO2.&lt;br/&gt; Combine with Equine Ringer&amp;rsquo;s Stock 1 to make complete &amp;ldquo;Ringer&amp;rsquo;s Solution&amp;rdquo;.</td></tr><tr><td>Pen/Strep solution</td><td>Gemini</td><td>400-109</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>P2636</td><td>0.5 mg/mL in 1x borate buffer</td></tr><tr><td>Prism software</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Protease</td><td>Sigma</td><td>P4630</td><td></td></tr><tr><td>Sodium bicarbonate solution</td><td>Sigma</td><td>S8761</td><td>7.5% stock solution</td></tr></tbody></table>

equine enteric glial culture and application to the study of a neural inflammatory mechanism in equine colic

Inflammatory postoperative conditions of equine colic (acute abdomen) contribute not only to increased client cost, patient discomfort, and hospitalization time, but in many cases, prove to be life-threatening. A unique population of intestinal cells, enteric glia, are increasingly acknowledged for their roles in sensing the gastrointestinal environment and communicating with surrounding cell types. Interactions between enteric glia and intestinal epithelia may prove critical in establishing how equine enteric glia can alter the mucosal barrier to modulate inflammation in health and colic.
To study this interaction, we present a method of establishing primary equine enteric glial cultures from equine jejunum and exposing the cultures to inflammatory conditions known to be present in colic. Primary enteric glial cultures were obtained from adult horses euthanized for reasons unrelated to colic. Intestinal villi and lamina propria were micro-dissected to expose the submucosa. The isolated submucosa underwent enzymatic digestion with collagenase, protease, and bovine serum albumin for 2-3 h. Next, mechanical digestion involving centrifugation, pipetting, and cell strainers (40-100 &#181;m), yielded a pellet used for plating on 0.05 mg/mL poly-L-lysine-coated wells at a concentration of ~400,000 cells/300 &#181;L of media.
Following confluence and first passage, the enteric glial cells were then exposed to equine recombinant IL-1&#946; (0, 10, 25 ng) for 24 h. To model epithelial-glial interactions at the time of colic, medium conditioned by either control or treated enteric glia was added directly to confluent equine jejunal monolayers while measuring transepithelial electrical resistance (TEER) using a dual-electrode EndOhm chamber. These data demonstrate just one of many potential impactful applications of equine enteric glial culture.

Microdissection of equine jejunum to the submucosal layer (Figure 1) with further enzymatic and mechanical digestion, could produce viable cell cultures of equine enteric glia. The cells demonstrated a pleomorphism with a dominance of spindle-shaped cells consistent with the enteric glia of other species (Figure 2A). The cultures were positive for the selective glial marker, glial fibrillary acidic protein (GFAP), with low fibroblast contamination (alpha SMA, <s...

Author Spotlight: Isolation and Characterization of Equine Submucosal Enteric Glia &#8212; Implications for Preventing Postoperative Complications in Colic Surgery

Enteric glia are becoming increasingly recognized for their roles in intestinal homeostasis and disease processes, including postoperative complications. Equine patients recovering from emergency exploratory laparotomy suffer from a high risk of inflammatory postoperative conditions, highlighting the importance of establishing repeatable equine enteric glial primary cell culture for study.

Equine Enteric Glial Culture and Application to the Study of a Neural Inflammatory Mechanism in Equine Colic

Inflammatory postoperative conditions of equine colic (acute abdomen) contribute not only to increased client cost, patient discomfort, and ...

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JoVE Journal

Medicine

246 Views.  NC State University. Paarden worden geconfronteerd met verwoestende inflammatoire postoperatieve aandoeningen na koliekchirurgie. In menselijk onderzoek winnen enterische gliacellen aan bekendheid als belangrijke sensoren van het darmmilieu, evenals beïnvloeders van ontstekingen. We wilden enterische gliacellen isoleren uit de dunne darm van paarden om hun rol bij het begrijpen en voorkomen van postoperatieve complicaties te beoordelen.We hebben een reproduceerbaar protoc...

Video: Auteur in de schijnwerpers: isolatie en karakterisering van submucosale darmglia bij paarden - implicaties voor het voorkomen van postoperatieve complicaties bij k

Immunostaining to Visualize Murine Enteric Nervous System Development

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Neural crest cells differentiate into neurons with markers specific for their neurotransmitter phenotype. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must ...

Developmental Biology

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Immunology and Infection

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Neuroscience

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell leukodystrophy (GLD) is unclear. This gap in knowledge has been hindered by the lack of an appropriate in vitro model for study. Herein, we describe a primary murine glial culture system in which treatment with psychosine results in multinucleation of microglia resembling the characteristic globoid cells found in GLD. Using this novel system, we defined the conditions and modes of analysis for study of globoid cells. The potential use of this model system was validated in our previous study, which identified a potential role for matrix metalloproteinase (MMP)-3 in GLD. This novel in vitro system may be a useful model in which to study the formation and function, but also the potential therapeutic manipulation, of these unique cells.

An In Vitro Model for the Study of Cellular Pathophysiology in Globoid Cell Leukodystrophy

Globoid cells are a defining pathological feature of Krabbe disease, a leukodystrophy currently lacking an effective long-term therapy. We have developed a cell culture model to study the innate biology and pathogenic potential of activated microglia and their transformation into globoid cells.

The precise function of multi-nucleated microglia, called globoid cells, that are uniquely abundant in the central nervous system of globoid cell ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Biology

Coculturing AdMSCs and Glia to Reduce Prion-Induced Inflammation

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments.
One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation.
A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Author Spotlight: Insight Into Advances in Prion Diseases Research

Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Equine Enteric Glial Culture and Application to the Study of a Neural Inflammatory Mechanism in Equine Colic

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