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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#8217;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 &#181;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 &#8216;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. 
			Combine with Equine Ringer&#8217;s Stock 2 to make complete &#8220;Ringer&#8217;s Solution&#8221;.</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 &#181;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&#946; 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&#8217;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. 
			Combine with Equine Ringer&#8217;s Stock 2 to make complete &#8220;Ringer&#8217;s Solution&#8221;.</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&#8217;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. 
			Combine with Equine Ringer&#8217;s Stock 2 to make complete &#8220;Ringer&#8217;s Solution&#8221;.</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&#8217;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. 
			Combine with Equine Ringer&#8217;s Stock 1 to make complete &#8220;Ringer&#8217;s Solution&#8221;.</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Thermo Fisher</td>
			<td>S271-10</td>
			<td>Component of Equine Ringer&#8217;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. 
			Combine with Equine Ringer&#8217;s Stock 2 to make complete &#8220;Ringer&#8217;s Solution&#8221;.</td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Thermo Fisher</td>
			<td>BP329-500</td>
			<td>Component of Equine Ringer&#8217;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. 
			Combine with Equine Ringer&#8217;s Stock 1 to make complete &#8220;Ringer&#8217;s Solution&#8221;.</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Thermo Fisher</td>
			<td>S637-212</td>
			<td>Component of Equine Ringer&#8217;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. 
			Combine with Equine Ringer&#8217;s Stock 1 to make complete &#8220;Ringer&#8217;s Solution&#8221;.</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.

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

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

Horses face devastating inflammatory post-operative conditions following Colic surgery. In human research, Enteric Glia cells are gaining notoriety as key sensors of the intestinal environment, as well as influencers of inflammation. We aimed to isolate Enteric Glia cells from equine small intestine in order to assess their role in understanding and preventing post operative complications.We have developed a reproducible protocol for isolating submucosal Enteric Glia cells from equine intestine. We were also able to demonstrate it's application to understanding the effect of inflammatory Enteric Glia on intestinal barrier function which may have important implications for future Colic post operative complication and intestinal disease research. Our lab will continue to focus on the relationship of Enteric Glia in inflammatory intestinal environments, as well as how the prevention of the loss of barrier function by anti-inflammatory biologics may be used to prevent post operative complications.

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...

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.

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

equine-enteric-glial-culture-application-to-study-neural-inflammatory

Research

JoVE Journal

Medicine

171 Views.  NC State University. Horses face devastating inflammatory post-operative conditions following Colic surgery. In human research, Enteric Glia cells are gaining notoriety as key sensors of the intestinal environment, as well as influencers of inflammation. We aimed to isolate Enteric Glia cells from equine small intestine in order to assess their role in understanding and preventing post operative complications.We have developed a reproducible protocol for isolating submucosal Enteric Glia cells from equine ...

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

Establishing Submucosal Equine Enteric Glial Primary Culture

To begin, coat the 24 well plates with the coating solution and incubate for two hours at 37 degrees Celsius, or overnight at room temperature. Wash the plates two times with sterile water and store them at room temperature until the cells are ready for plating. Now take a section of the horse jejunum and place it in cold ringer solution on ice.Remove a 10 centimeter portion from the jejunum and open it at the anti-mesenteric border, positioning non-lymphoid regions at the center. Then trim the portion into an approximately seven by seven centimeter section, and rinse the tissue well in fresh ringer solution prior to dissection. Now pin the tissue on a silicone elastomer coated Petri dish in cold ringer solution, or PBS, with the mucosal side up, while stretching it as much as possible.Using curved forceps, remove the intestinal villi, and then remove the lamina propria layer from approximately five by five centimeter non-lymphoid region. Next, with microdissection scissors, remove the submucosa in one sheet by freeing it at one corner. Observe the natural separation while peeling away the submucosa from the inner circular muscle layer.Then mince the collected submucosal layer into two to five millimeter pieces. Place them into a 50 milliliter conical tube, containing five milliliters of orgono without FBS, collagenase, protease, and BSA. Incubate the tissue for two to three hours at 37 degrees Celsius for enzymatic digestion.After incubation, add 10 milliliters of room temperature orgono with FBS to stop the enzyme action. Pipette the tissue mixture up and down 15 times using a 10 milliliter serological pipette to dissociate the cells from the tissue. Then centrifuge the mixture at 3000 G for three minutes at room temperature.Afterward, discard the supernatant and resuspend the pellet in 10 milliliters of room temperature PBS by pipetting the solution up and down 15 times with a 10 milliliter serological pipette. Centrifuge the conical tube at 3000 G for three minutes at room temperature. After discarding the supernatant, resuspend the pellet in 10 milliliters of room temperature orgono with FBS by pipetting up and down 15 times.Next, filter the cells sequentially through a 100 micrometer, 70 micrometer, and 40 micrometer pore size cell strainer. After centrifuging the cells and discarding the supernatant, resuspend the pellet in one milliliter of orgono with FBS. Then stain the cells with trypan blue to identify live cells and count them using a hemocytometer.Now seed approximately 400, 000 cells in 300 microliters of orgono with FBS in each well of a 24 well plate. Add five microliters of N2, five microliters of G5, and 10 microliters of B27 to each well. Place the plate in a 37 degrees Celsius incubator with 5%carbon dioxide to allow cell adhesion.When cells from the first passage reach 70 to 80%confluence, expose the wells to zero, 10, and 25 nanograms of interleukin-1 beta for 24 hours. Spindle-shaped cells consistent with enteric glial morphology were observed in the cultures, with pleomorphism and minimal contamination from other cell types.

Equine Organoid Culture for Production of Enterocyte Monolayers and their Response to Inflammatory Cytokine and Glial Products 

To begin, coat the Transwell inserts with 42 microliters of 0.05%Matrigel. Incubate for one hour at 37 degrees Celsius to allow Matrigel to set. Aspirate the extra medium and dry the Transwells for 30 minutes uncovered.Then add 200 microliters of DMEM F12 medium to each insert and store at 37 degrees Celsius until use. Wash the Matrigel patties containing the organoids with PBS. Expose them to 500 microliters of cell recovery while on ice, and pipette the mixture repeatedly to ensure a full collection of cells in a 15 milliliter tube.Then centrifuge the tube at 300g for five minutes at four degrees Celsius. Remove the supernatant and resuspend the cell pellet in the target volume of intestinal epithelial stem cell or IESC medium. Now, pre-coat a one milliliter syringe in IESC medium, aspirate the cell suspension through a 16-gauge needle.Then replace the 16-gauge needle with a 28-gauge needle and eject the suspension to promote separation of the organoids. Next, place 200 microliters of the resulting cell suspension into the apical side of the coated Transwells. Add 500 microliters of IESC medium and growth factors to the basal side.Use a dual electrode TEER measuring chamber to quantify the TEER across the monolayers. Fill the culture cup with 1.5 milliliters of IESC media and ensure exactly 200 microliters of media is present in the Transwell insert, so that the fluid levels match during measurement. Expose the basolateral aspect of the Transwell inserts to either inflammatory cytokines of interleukin-1 beta or glial products from cultures exposed to interleukin-1 beta.Measure the TEER every 10 to 15 minutes for 45 minutes by placing each Transwell into the culture cup. Basolateral exposure to media from glial cultures treated with 10 and 25 nanograms of interleukin-1 beta significantly increased epithelial barrier permeability. No significant increase in epithelial permeability was observed after exposure to equine interleukin-6 across various concentrations.