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.

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 ...

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

Research

JoVE Journal

Medicine

221 Views.  NC State University. 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. 

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

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Studying the Hypothalamic Insulin Signal to Peripheral Glucose Intolerance with a Continuous Drug Infusion System into the Mouse Brain

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism.
The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals.
After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.

This protocol studies the role of chemokine (C-C motif) ligand 5 (CCL5) in the hypothalamus by delivering an antagonist, MetCCL5, into the mouse brain using a micro-osmotic pump brain infusion system. This transient inhibition of CCL5 activity interrupted hypothalamic insulin signaling, leading to glucose intolerance and peripheral systemic insulin sensitivity.

Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues ...

Nerve Stimulator-guided Injection of Autologous Stem Cells Near the Equine Left Recurrent Laryngeal Nerve

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent laryngeal nerve shows lesions of demyelination. The benefit of applying stem cells to demyelinated nerves has been demonstrated in various animal models. The aim of the study was to test the feasibility and safety of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to the left recurrent laryngeal nerve in healthy horses by using an electrical nerve stimulator.
Muscle-derived stems cell are obtained from five healthy Standardbred horses by sampling 20 mg of muscle tissue with a semi-automatic 14 G biopsy needle from the triceps muscle. Movements of the larynx are monitored via upper-airway video endoscopy. The left recurrent laryngeal nerve is approached with an insulated nerve block needle. Nerve stimulation is applied, starting at 2 mA, and the successful abduction of the left arytenoid is monitored. The stimulation intensity is reduced progressively. When a loss of the motor response is observed at 0.5 mA, 107 autologous muscle-derived stem cells are injected. Two examiners, who are blinded to the time point, score the laryngeal function of the horses prior to the treatment and at day 1, day 7, and day 28 after the injection of the cells. In a sixth horse, 1 mL of 2% lidocaine is injected to further confirm the correct positioning of the needle. This leads to a temporary paralysis of the left arytenoid cartilage.
This study proves that the recurrent laryngeal nerve can be approached with the help of an electrical nerve stimulator and that the electrical stimulation of the nerve is well tolerated by the horses. No modification of the laryngeal function was observed in any of the horses after the injection of the stem cells. Further studies should be conducted to describe the effects of a peri-neuronal injection of autologous muscle-derived mesenchymal stem cells to horses suffering from RLN.

Here, we present a protocol to inject autologous muscle-derived stem cells in the proximity to the recurrent laryngeal nerve with the help of an electrical nerve stimulator. This novel technique may become useful for the treatment of equine recurrent laryngeal neuropathy.

Recurrent laryngeal neuropathy (RLN) commonly affects horses and is characterized by abnormal respiratory sounds and exercise intolerance. The recurrent ...

Precision Implementation of Minimal Erythema Dose (MED) Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary to produce erythema (inflammatory reddening) on the surface of the skin. In this context, the MED is regarded as a key factor in determining starting doses for UV phototherapy for common skin conditions such as psoriasis and eczema. In research settings, MED testing also has potential to be a powerful tool for assessing within- and between-persons variation in inflammatory responses. However, MED testing has not been widely adopted for use in research settings, likely owing to a lack of published guidelines, which is a barrier to obtaining reproducible results from this assay. Also, protocols and equipment for establishing MED vary widely, making it difficult to compare results across laboratories. Here, we describe a precise and reproducible method to induce and measure superficial erythema using newly designed protocols and methods that can easily be adapted to other equipment and laboratory environments. The method described here includes detail on procedures that will allow extrapolation of a standardized dosage schedule to other equipment so that this protocol can be adapted to any UV radiation source.

Precision Implementation of Minimal Erythema Dose MED Testing to Assess Individual Variation in Human Inflammatory Response

Minimal erythema dose (MED) testing is used to establish dosage schedules for ultraviolet radiation phototherapy. It can assess individual variation in inflammatory response but lacks methodology for achieving reproducible results. Here, we present a precision implementation of MED and demonstrate its ability to capture individual variation in inflammatory response.

Minimal erythema dose (MED) testing is frequently used in clinical settings for determining the smallest amount of ultraviolet (UV) irradiation necessary ...

Endoscopy Guided Photoablation of Endometrial Cysts using a 980 nm Laser with a Contact Fiber in Mares

In mares, endometrial cysts are associated with endometriosis and can cause maternal recognition failure or compromise and delay pregnancy diagnoses. Historical treatments were invasive and had adverse effects on the endometrium. Hysteroscopically guided laser therapy is easy and effective for endometrial cysts resection, with no deleterious effects for the endometrium.
A 110 cm long and 1.0 cm wide endoscope is sterilely introduced in the uterus through the open cervix of an estrous mare after vulvar cleaning. The uterus is slowly infused with less than 1 L of physiologic solution and the laser fiber is inserted in the biopsy canal of the endoscope. Cysts are then cauterized with the 980 nm diode laser with a contact fiber set at 20&#8210;2 5W in continuous mode. Each cyst is punctured&#160;until complete voiding of the cyst and shrinking of the cyst wall around the fiber. Uterine lavages with sterile saline solution are performed directly after the surgery and for one or two days as non-inflammatory fluid can be observed.
This procedure is easy and quickly performed, with no obvious deleterious effects. Cysts resection makes ultrasound pregnancy diagnosis easier and, in some cases, could restore proper embryo migration in the uterine horns between day 6.5 and 17. However, this treatment does not improve the underlying histological lesions related to endometriosis. These considerations should be clearly expressed to the breeder before this procedure.

The aim of this report is to describe the photoablation of endometrial cysts in the mare using an endoscopic laser device, and to discuss the interest and limitations of this new approach to deal with low fertility issues.

In mares, endometrial cysts are associated with endometriosis and can cause maternal recognition failure or compromise and delay pregnancy diagnoses. ...

Application of Optical Coherence Tomography to a Mouse Model of Retinopathy

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images from which the retinal thickness can be calculated. Although OCT is widely used in clinical practice, its application in basic research is not as prevalent, especially in small animals such as mice. Because of the small size of their eyeballs, it is challenging to conduct fundus imaging examinations in mice. Therefore, a specialized retinal imaging system is required to accommodate OCT imaging on small animals. This article demonstrates a small-animal-specific system for OCT examination procedures and a detailed method for image analysis. The results of retinal OCT examination of very-low-density lipoprotein receptor (Vldlr) knockout mice and C57BL/6J mice are presented. The OCT images of C57BL/6J mice showed retinal layers, while those of Vldlr knockout mice showed subretinal neovascularization and retinal thinning. In summary, OCT examination could facilitate the noninvasive detection and measurement of retinopathy in mouse models.

Here, we describe an in vivo imaging technique using optical coherence tomography to facilitate the diagnosis and quantitative measurement of retinopathy in mice.

Optical coherence tomography (OCT) offers a noninvasive method for the diagnosis of retinopathy. The OCT machine can capture retinal crosssectional images ...