This content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. HC is supported by grant P20GM134973 from the National Institutes of Health. KB is supported by a Children&#39;s Hospital Foundation (CHF) and Presbyterian Health Foundation (PHF) grant. Live-cell imaging services provided by the Cancer Functional Genomics core were supported partly by the National Institute of General Medical Sciences Grant P20GM103639 and National Cancer Institute Grant P30CA225520 of the National Institutes of Health, awarded to the University of Oklahoma Health Sciences Center Stephenson Cancer Center.

All animal procedures in this study were approved by the University of Oklahoma Health Sciences Center Institutional Animal Care and Use Committee. Following institutional approval, fetal small intestine convenience samples from a preterm non-human primate (NHP, 90% gestation, olive baboon, Papio anubis) were obtained following euthanasia for a separate study (Protocol #101523-16-039-I)20.
1. Establishment of preterm non-human primate 3D intestinal enteroids
<ol>
	<li>Media preparation 
	NOTE: Media can be prepared up to 1 week in advance of crypt isolation following standard aseptic technique. Penicillin/streptomycin (final concentration 1%) may be used in place of the broad-spectrum antibiotics for primary cells if desired.
	<ol>
		<li>Prepare human organoid growth media + Y-27632 (HOGMY) by mixing 50 mL of organoid growth medium human basal medium with 50 mL of organoid supplement (Table of Materials). Add 200 &#181;L of the broad-spectrum antibiotics (final concentration 100 &#181;g/mL) (Table of Materials) and Y-27632 (to a final concentration of 10 &#181;M). Warm on the benchtop to room temperature.</li>
		<li>Prepare human organoid growth media (HOGM) by mixing 50 mL of organoid growth medium human basal medium with 50 mL of organoid supplement. Add 200 &#181;L of the broad-spectrum antibiotics (100 &#181;g/mL). Warm on the benchtop to room temperature. 
		NOTE: HOGM will be used when changing media beyond the first 2-3 days post-passage.</li>
		<li>Chill 100 mL of 1x phosphate-buffered saline (PBS) without calcium or magnesium until ice-cold.</li>
		<li>Prepare 50 mL of wash buffer by combining 1x PBS without calcium or magnesium with 0.1% bovine serum albumin (BSA). Chill it until ice-cold.</li>
		<li>Prepare DMEM-F12 wash buffer by adding 1 mL of the broad-spectrum antibiotics (100 &#181;g/mL) to 500 mL of DMEM/F12 (Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Ham&#39;s Mixture F12 + 15 mM HEPES buffer). Keep it ice-cold.</li>
	</ol>
	</li>
	<li>Crypt isolation and plating 
	NOTE: Extracellular matrix (ECM)-based hydrogel must remain on the ice during the entire procedure. This procedure assumes enough tissue to populate six ECM-based hydrogel domes. If a different density of crypts is harvested, the dome number should be altered accordingly.
	<ol>
		<li>Thaw 150 &#181;L of growth factor reduced (GFR) basement membrane extract (BME) (phenol red-free) overnight on the ice at 2-8 &#176;C.</li>
		<li>Incubate a 24-well, flat-bottom, tissue culture-treated polystyrene plate in an incubator at 37 &#176;C and 5% CO2 for a minimum of 30 min before use.</li>
		<li>Following NHP euthanasia and tissue collection, flush the ileum segment of debris with a 1,000 &#181;L pipette tip using ice-cold 1x PBS in a disposable Petri dish. 
		NOTE: Fresh tissue must be harvested quickly and kept ice-cold to harvest healthy enteroids.</li>
		<li>Cut the ileum longitudinally along the entire intestine, and wash with ice-cold PBS 3x. Mince the tissue into small fragments (approximately 2 mm in length) with sterile scissors over a 50 mL conical tube containing 15 mL ice-cold PBS, starting from the portion of the ileal segment closest to the tube.</li>
		<li>Use a 10 mL serological pipette to disrupt the crypts by pipetting up and down 5-10x. Let the tissue segments settle to the bottom of the tube, then remove the supernatant and replace it with fresh, ice-cold PBS. Repeat this process a minimum of 7x-10x until the supernatant is clear and free of debris.</li>
		<li>Once clear, remove the supernatant and resuspend the crypts in 25 mL of cell dissociation reagent. Place the tube on a rocking platform at 20 rpm for 15 min at room temperature.</li>
		<li>Remove the tube from the rocker, allow the cells to settle for approximately 30 s to 1 min, and remove the supernatant. Add 10 mL of ice-cold wash buffer. Pipette up and down with a p1000 pipette to further dissociate into single crypts.</li>
		<li>Filter the tissue through a 70 &#181;m cell strainer into a new 50 mL conical tube. Rinse the original 50 mL conical tube with 10 mL of ice-cold wash buffer and filter through the 70 &#181;m strainer to ensure all the crypts have been removed from the original tube. Repeat this washing of tissue for a total of 4x rinses.</li>
		<li>Centrifuge the filtrate at 300 x g for 5 min at 4 &#176;C and remove the supernatant. Add 600 &#181;L of HOGMY and disrupt the pellet by pipetting up and down with a 200 &#181;L pipette. Place the tube on ice until the solution becomes ice-cold. 
		NOTE: If the solution is not ice-cold, the dome will not maintain the proper structure when plated. Avoid introducing bubbles when pipetting ECM-based hydrogel&#160;domes, as bubbles will prevent proper adherence to the plate bottom.</li>
		<li>Add 600 &#181;L of ice-cold ECM-based hydrogel to suspend the cell pellet and mix well with a 200 &#181;L pipette.</li>
		<li>Remove the 24-well plate from the incubator and place it on a 37 &#176;C plate warmer. Pipette 50 &#181;L of ice-cold HOGMY/ECM-based hydrogel mixture into the center of 24 wells of a 24-well culture plate (50 &#181;L per dome).</li>
		<li>Once plated, let the domes sit on a 37 &#176;C plate warmer for 1 min to allow adherence to the bottom of the plate. Carefully invert the plate, ensure no condensation has accumulated on the bottom of the plate, and place the inverted plate in the incubator at 37 &#176;C and 5% CO2.</li>
		<li>After 15 min, turn the plate right-side-up and pipette 750 &#181;L of room-temperature HOGMY into each well. 
		NOTE: Pipette media slowly onto the sidewall of wells, being careful not to disrupt the newly formed 3D ECM-based hydrogel dome.</li>
		<li>Once all the wells have been filled with media, place the plate in the incubator and change the media every 3 days with 750 &#181;L of HOGM. Passage enteroids every 7-10 days.</li>
	</ol>
	</li>
	<li>Intestinal enteroid passaging 
	NOTE: The following protocol is for one 24-well culture plate containing NHP enteroids at a split rate of 1:2.
	<ol>
		<li>Remove the enteroid media from each well, being careful not to disrupt the ECM dome containing enteroids, add 500 &#181;L of ice-cold cell dissociation reagent to each well using a 1,000 &#181;L pipette, and incubate at room temperature for 1 min.</li>
		<li>Manually disrupt the ECM by pipetting up and down 8-12x. Transfer the contents of 12 wells to one 15 mL conical tube.</li>
		<li>Once all the solution has been collected from the 24-well culture plate, add 250 &#181;L of cell disruption reagent to each well to ensure all the NHP enteroids have been removed from each well, adding this last rinse to the existing 15 mL conical tubes.</li>
		<li>Place on a rocking platform at 40 rpm for 15 min at room temperature (20 &#176;C). Alternatively, manually shake the tubes for 15 min at room temperature.</li>
		<li>After 15 min, centrifuge at 300 x g for 5 min at 4 &#176;C. Remove the supernatant from the 15 mL tubes until only the pellet remains.</li>
		<li>Add 8 mL of ice-cold DMEM + broad-spectrum antibiotics to one 15 mL conical tube, making sure the pellet is sufficiently disrupted. Centrifuge at 300 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
		<li>Add 600 &#181;L of HOGMY and disrupt the pellet by pipetting up and down with a 1,000 &#181;L pipette. Place the tube on ice until the solution becomes ice-cold. Once the solution is ice-cold, add 600 &#181;L of ice-cold ECM to suspend the cell pellet and mix well with a 1,000 &#181;L pipette. Repeat steps 1.3.2.-1.3.7. for the remaining 12 wells.
		<ol>
			<li>For the remaining steps, refer to steps 1.2.11.-1.2.16.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Establishment of enteroid monolayer and scratch wound assay
<ol>
	<li>Media and treatment preparation
	<ol>
		<li>Chill 5 mL of PBS without calcium or magnesium until ice-cold.</li>
		<li>Prepare DMEM-F12 wash buffer by adding 1 mL of the broad-spectrum antibiotics (100 &#181;g/mL) to 500 mL of DMEM/F12 (Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Ham&#39;s Mixture F12 + 15 mM HEPES buffer). Keep ice-cold.</li>
		<li>Prepare 100 mL of HOGMY, as described in step 1.1.1., and warm it on the benchtop to room temperature.</li>
		<li>Warm 25 mL of 0.25% Trypsin-ethylenediaminetetraacetic acid (EDTA) in a 37 &#176;C water bath.</li>
		<li>Prepare 50 &#181;g/mL, 100 &#181;g/mL, and 200 &#181;g/mL concentrations of HA35 for cell treatment, using sterile PBS without calcium or magnesium as a solvent. The final dilution of HA35 will use HOMGY as the solvent. 
		NOTE: HA35 preparation may require several dilutions, as HA35 often comes out of solution at a concentration greater than 5 mg/mL.</li>
	</ol>
	</li>
	<li>Enteroid monolayer formation 
	NOTE: The following procedure provides volumes sufficient for the generation of one 24-well plate of enteroid monolayers. Adjust the volumes for the desired number of monolayers.
	<ol>
		<li>Thaw 96 &#181;L of ECM-based hydrogel BME (phenol red-free) overnight on ice at 2-8 &#176;C. Dilute ECM-based hydrogel in ice-cold PBS without calcium or magnesium at a ratio of 1:50. Coat each well of a 24-well tissue culture plate with 200 &#181;L of ice-cold diluted ECM-based hydrogel.</li>
		<li>Incubate the ECM-based hydrogel-coated plate for a minimum of 1 h at 37 &#176;C and 5% CO2. Aspirate media from the 24-well culture plate containing the 3D enteroids destined for monolayers and replace with 500 &#181;L of cell dissociation reagent/well.</li>
		<li>Manually disrupt ECM-based hydrogel domes by pipetting up and down 8x-12x and transfer the contents of 12 wells to a 15 mL conical tube.</li>
		<li>Wash the 12 empty wells with 250 &#181;L of cell dissociation reagent to ensure all the enteroids have been removed and transfer the contents to a 15 mL conical tube. Repeat step 2.2.5. and step 2.2.6. for the remaining 12 wells using a second 15 mL conical tube.</li>
		<li>Centrifuge the conical tubes at 300 x g for 5 min at 4 &#176;C and discard the supernatant. Add 10 mL of ice-cold DMEM-F12 wash buffer to 15 mL conical tubes and suspend the enteroid pellets by inverting the tubes. Centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
		<li>Remove the supernatant and resuspend the cell pellets in 12 mL of 37 &#176;C Trypsin-EDTA. Place the tubes in a 37 &#176;C water bath for 10 min or until the cells appear soluble. Add 3 mL of DMEM-F12 wash buffer to each tube to dilute Trypsin-EDTA, mix by inverting the tubes, and place on ice.</li>
		<li>Filter the dissociated enteroids through a 37 &#181;M mesh cell strainer, collecting the contents into clean 15 mL conical tubes. Centrifuge the tubes at 300 x g for 5 min at 4 &#176;C.</li>
		<li>While the cells are pelleting in the centrifuge, remove the ECM-based hydrogel-coated plate from the incubator and aspirate any excess ECM-based hydrogel solution without scratching the coated surface or letting the plate completely dry.</li>
		<li>Remove the supernatant from the conical tubes and resuspend the cell pellets with 6 mL of HOMGY. Add 500 &#181;L of the combined 12 mL HOMGY cell suspension into each well of the 24-well plate coated with ECM-based hydrogel at a seeding density of roughly 3 x 105 cells/well. Swirl the plate with a lid on to ensure an even distribution of cells within the wells.</li>
		<li>Incubate the plate at 37 &#176;C and 5% CO2, exchanging HOMGY media every 2 days until the monolayers reach &#62;90% confluency.</li>
	</ol>
	</li>
	<li>Monolayer treatment and scratch wound assay
	<ol>
		<li>Once the monolayers reach &#62;90% confluency, aspirate media to remove any cells failing to attach. Treat cells with 500 &#181;L of HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or PBS, dissolved in HOMGY, for 24 h.</li>
		<li>After 24 h, remove the media, being careful not to disrupt the surface of the monolayer. Using a 200 &#181;L pipette tip, make a linear scratch across the full surface diameter of the monolayer in each well, being careful not to let monolayers dry.</li>
		<li>Wash the scratched monolayers 1x with 500 &#181;L of 1x PBS. Add 500 &#181;L of HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or PBS dissolved in HOMGY and transfer the plate to the live-cell analysis instrument (Table of Materials) within a 37 &#176;C and 5% CO2 incubator.</li>
		<li>Under the Schedule icon in the live-cell analysis software (Table of Materials), click on the Launch Add New Vessel Wizard icon and set the scanning frequency to scan on a schedule. Create a new Whole Well vessel under Scan Type, specify the scan settings (i.e., Phase for Image Channels and 4x for Objective), and click on Next.</li>
		<li>Select the plate manufacturer and catalog number from the provided list. Under the Schedule icon, choose the plus symbol where the plate will be placed within the live-cell analysis instrument. Select the&#160;desired Scan Pattern, and then create a plate map by selecting&#160;&#34;+&#34; on the&#160;Plate Layout&#160;page. Select Defer Analysis Until Later, and then set the Scanning Schedule for every 4 h for 24 h and verify acquisition settings under the Vessel Wizard Summary page. Finally, select&#160;Add to Schedule.</li>
		<li>Once the 24 assay is complete, under the View icon, double-click Vessel Name, and export images and movies. Select wells and a series of images to display and export as a JPEG. Analyze images for percent wound healing using the Wound Healing Size Plugin for ImageJ21, as per step 2.4. below.</li>
	</ol>
	</li>
	<li>Scratch wound analysis
	<ol>
		<li>Open ImageJ software. Upload the scratch wound assay images (.TIFF or .JPG).</li>
		<li>Select Image &#62; Type &#62; 8-bit to change the image from 24-bit RGB to 8-bit. Install the Wound Healing Size Plugin by selecting Plugins &#62; Macros &#62; Install &#62; Wound Healing Size Plugin.</li>
		<li>Rotate the image so the scratch is vertical and initialize the Wound Healing Size Tool plugin.</li>
		<li>Select the plugin parameters in the dialog box to best fit the scratch wound. Set the value of the parameters for wound healing size options as follows: Variance Window Radius at 20, Threshold value at 100, and Percentage of Saturated Pixels at 0.001, and choose Yes for Set Scale Global. Finalize the selection by clicking on the OK button. Verify if the selected area is the wound area. The above parameters may require standardization from lab to lab.</li>
		<li>Repeat steps 2.4.2.-2.4.4. for the remaining time points for each scratch.</li>
		<li>Calculate the percentage of scratch area wound healing of migrating or proliferating cells over 24 h as follows: 
		%&#160;Wound Healing = <img alt="Equation 1" src="/files/ftp_upload/63758/63758eq01.jpg" style="margin: auto;" /> 
		where T0 = % Area at time 0 h and Tc = % Area at 0 h, 4 h, 12 h, or 24 h.</li>
	</ol>
	</li>
</ol>

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P., 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=Multifunctional+role+of+35+kilodalton+hyaluronan+in+promoting+defense+of+the+intestinal+epithelium.">Multifunctional role of 35 kilodalton hyaluronan in promoting defense of the intestinal epithelium.</a> The Journal of Histochemistry and Cytochemistry. 66 (4), 273-287 (2018).</li><li>Fraser, J. R. E., Laurent, T. C., Laurent, U. B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan:+Its+nature,+distribution,+functions+and+turnover.">Hyaluronan: Its nature, distribution, functions and turnover.</a> Journal of Internal Medicine. 242 (1), 27-33 (1997).</li><li>Kim, Y., 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=Hyaluronan+35kDa+treatment+protects+mice+from+Citrobacter+rodentium+infection+and+induces+epithelial+tight+junction+protein+ZO-1+in+vivo.">Hyaluronan 35kDa treatment protects mice from Citrobacter rodentium infection and induces epithelial tight junction protein ZO-1 in vivo.</a> Matrix Biology. 62, 28-39 (2017).</li><li>Prehm, P., Schumacher, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+hyaluronan+export+from+human+fibroblasts+by+inhibitors+of+multidrug+resistance+transporters.">Inhibition of hyaluronan export from human fibroblasts by inhibitors of multidrug resistance transporters.</a> Biochemical Pharmacology. 68 (7), 1401-1410 (2004).</li><li>Stenson, W. F., Ciorba, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonmicrobial+activation+of+TLRs+controls+intestinal+growth,+wound+repair,+and+radioprotection.">Nonmicrobial activation of TLRs controls intestinal growth, wound repair, and radioprotection.</a> Frontiers in Immunology. 11 (3591), 617510 (2021).</li><li>Riehl, T. E., Ee, X., Stenson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronic+acid+regulates+normal+intestinal+and+colonic+growth+in+mice.">Hyaluronic acid regulates normal intestinal and colonic growth in mice.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 303 (3), 377-388 (2012).</li><li>Riehl, T. E., Santhanam, S., Foster, L., Ciorba, M., Stenson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD44+and+TLR4+mediate+hyaluronic+acid+regulation+of+Lgr5++stem+cell+proliferation,+crypt+fission,+and+intestinal+growth+in+postnatal+and+adult+mice.">CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 309 (11), 874-887 (2015).</li><li>Hill, D. R., Kessler, S. P., Rho, H. K., Cowman, M. K., de la Motte, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific-sized+hyaluronan+fragments+promote+expression+of+human+beta-defensin+2+in+intestinal+epithelium.">Specific-sized hyaluronan fragments promote expression of human beta-defensin 2 in intestinal epithelium.</a> Journal of Biological Chemistry. 287 (36), 30610-30624 (2012).</li><li>Fernando, E. H., Gordon, M. H., Beck, P. L., MacNaughton, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+intestinal+epithelial+wound+healing+through+protease-activated+receptor-2+activation+in+Caco2+cells.">Inhibition of intestinal epithelial wound healing through protease-activated receptor-2 activation in Caco2 cells.</a> Journal of Pharmacology and Experimental Therapeutics. 367 (2), 382-392 (2018).</li><li>Nyegaard, S., Christensen, B., Rasmussen, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+method+for+accurate+quantification+of+cell+migration+using+human+small+intestine+cells.">An optimized method for accurate quantification of cell migration using human small intestine cells.</a> Metabolic Engineering Communications. 3, 76-83 (2016).</li><li>Roodsant, T., 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=A+human+2D+primary+organoid-derived+epithelial+monolayer+model+to+study+host-pathogen+interaction+in+the+small+intestine.">A human 2D primary organoid-derived epithelial monolayer model to study host-pathogen interaction in the small intestine.</a> Frontiers in Cellular and Infection Microbiology. 10, 272 (2020).</li><li>Singh, A., Poling, H. M., Spence, J. R., Wells, J. M., Helmrath, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastrointestinal+organoids:+a+next-generation+tool+for+modeling+human+development.">Gastrointestinal organoids: a next-generation tool for modeling human development.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 319 (3), 375-381 (2020).</li><li>Foulke-Abel, J., 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=Human+enteroids+as+an+ex-vivo+model+of+host-pathogen+interactions+in+the+gastrointestinal+tract.">Human enteroids as an ex-vivo model of host-pathogen interactions in the gastrointestinal tract.</a> Experimental Biology and Medicine. 239 (9), 1124-1134 (2014).</li><li>Foulke-Abel, J., 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=Human+enteroids+as+a+model+of+upper+small+intestinal+ion+transport+physiology+and+pathophysiology.">Human enteroids as a model of upper small intestinal ion transport physiology and pathophysiology.</a> Gastroenterology. 150 (3), 638-649 (2016).</li></ol>

The authors have nothing to disclose and no conflicts of interest.

The gastrointestinal tract of a preterm infant is under continual regenerative pressure from repeated exposures to environmental insults associated with dysbiosis, inflammatory bacterial metabolites and toxins, and intermittent hypoxia23,24. Unfortunately, the intestinal epithelium of the preterm infant is unable to rapidly establish functional integrity23, resulting in barrier dysfunction, increased intestinal permeability, and, in severe...

Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal emergencies in preterm infants1. The disease is characterized by severe intestinal inflammation that can rapidly deteriorate to intestinal necrosis, sepsis, and potentially death. Although the etiology is unclear, evidence suggests NEC is multifactorial and the result of a complex interaction of feeding, abnormal bacterial colonization, and an immature intestinal epithelium2,3. Preterm infants have increased intestinal permeability, abnormal bacterial colonization, and low enterocyte regenerative capacity4,5, increasing their risk for intestinal barrier dysfunction, bacterial translocation, and NEC development. Therefore, identifying strategies or interventions to accelerate intestinal epithelial maturation and promote regeneration or healing of the intestinal epithelium is critical in preventing this deadly disease.
Studies have demonstrated that human milk (HM) is protective against NEC in preterm infants6,7,8,9,10,11. Both human and animal studies have shown that bovine-based formula increases intestinal permeability and is directly toxic to intestinal epithelial cells2,12. Although not fully elucidated, evidence suggests the protective effects of HM are mediated through bioactive components such as lactoferrin, immunoglobulin A (IgA), and HM oligosaccharides13. HM is also rich in hyaluronan (HA), a uniquely nonsulfated glycosaminoglycan with repeating D-glucuronic acid and N-acetyl-D-glucosamine disaccharides14,15. Importantly, we have shown that oral 35 kDa HA (HA35), an HM HA mimic, attenuates the severity of intestinal injury, prevents bacterial translocation, and decreases mortality in a murine NEC-like intestinal injury model16,17.
Here, the effects of HA35 on intestinal healing and regeneration in vitro are further investigated. Currently, the most widely used in vitro assay for intestinal wounding and repair is a scratch wound assay performed in colorectal cancer (CRC) cell monolayers. The physiological relevance of such a model to the preterm infant intestine is limited, as wound repair of CRC cells relies heavily upon the highly proliferative nature of cancer cells rather than stem cell-driven repair processes18. To overcome this limitation, the establishment of a 2D enteroid scratch wound model, including the procedure of isolating and maintaining primary stem cell-derived small intestinal enteroids from preterm non-human primates (NHP), is described here. Given preterm NEC is most often reported in the distal small intestine, the use of primary epithelial cell organoids in a model of intestinal damage and repair provides a more physiologically translatable in vitro model compared with existing models utilizing traditional colorectal monolayers18,19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 mL Serological Pipet</td>
			<td>Fisher Scientific</td>
			<td>13-675-49</td>
			<td></td>
		</tr>
		<tr>
			<td>100x21mm Dish, Nunclon Delta</td>
			<td>ThermoFisher Scientific</td>
			<td>172931</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical tube</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well, TC-Treated, Flat Bottom Plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#181;M Reversible Cell Strainer</td>
			<td>STEMCELL Technologies</td>
			<td>27215</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical tube</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#181;m Sterile Cell Strainers</td>
			<td>Fisher Scientific</td>
			<td>FB22-363-548</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine (BSA)</td>
			<td>VWR</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo 3D Cell Viability Assay</td>
			<td>Promega</td>
			<td>G9681</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Ham&#39;s Mixture F-12 (DMEM-F12) with 15 mM HEPES buffer</td>
			<td>STEMCELL Technologies</td>
			<td>36254</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentle Cell Dissociation Reagent</td>
			<td>STEMCELL Technologies</td>
			<td>100-0485</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td>imagej.nih.gov/ij/</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte S3 Live-Cell Analysis Instrument</td>
			<td>Sartorius</td>
			<td>4647</td>
			<td></td>
		</tr>
		<tr>
			<td>Incucyte Scratch Wound Analysis Software Module</td>
			<td>Sartorius</td>
			<td>9600-0012</td>
			<td></td>
		</tr>
		<tr>
			<td>IntestiCult Organoid Growth Medium (Human)</td>
			<td>STEMCELL Technologies</td>
			<td>06010</td>
			<td>This is HOGMY, but without the Y-27632 or antibiotics. Also used as base for HOGM, but then only missing the antibiotics.</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides from Escherichia coli O111:B4, purified by gel filtration chromatography</td>
			<td>Millipore Sigma</td>
			<td>L3012-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td>
			<td>ThermoFisher Scientific</td>
			<td>136101</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-Buffered Saline), 1X [-] Calcium, Magnesium, pH 7.4</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td>This is broad-spectrum antibiotics</td>
		</tr>
		<tr>
			<td>Sodium Hyaluronate, Research Grade, HA20K</td>
			<td>Lifecore Biomedical</td>
			<td>HA20K-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>Company: Bio-Rad</td>
			<td>1450102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 1X, 0.25% Trypsin</td>
			<td>Fisher Scientific</td>
			<td>MT25053CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>STEMCELL Technologies</td>
			<td>72302</td>
			<td></td>
		</tr>
	</tbody>
</table>

effect of hyaluronic acid 35 kda on an in vitro model of preterm small intestinal injury and healing using enteroid-derived monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. Here, we describe a protocol to generate a two-dimensional (2D) monolayer from three-dimensional (3D) non-human primate enteroids derived from intestinal crypts of the terminal ileum. These enteroid-derived monolayers were then utilized in an in vitro scratch wound assay to test the ability of hyaluronan 35 kDa (HA35), a human milk HA mimic, to promote cell migration and proliferation along the epithelial wound edge. After the monolayers were grown to confluency, they were manually scratched and treated with HA35 (50 &#181;g/mL, 100 &#181;g/mL, 200 &#181;g/mL) or control (PBS). Cell migration and proliferation into the gap were imaged using a transmitted-light microscope equipped for live-cell imaging. Wound closure was quantified as percent wound healing using the Wound Healing Size Plugin in ImageJ.&#160;The scratch area and rate of cell migration and the percentage of wound closure were measured over 24 h. HA35 in vitro accelerates wound healing in small intestinal enteroid monolayers, likely through a combination of cell proliferation at the wound edge and migration to the wound area. These methods can potentially be used as a model to explore intestinal regeneration in the preterm human small intestine.

The effects of HA on tissue repair and wound healing in various tissues and organs are well-documented; however, the specific effects of HA with a molecular weight of 35 kDa on fetal or neonatal small intestinal healing and regeneration are currently unknown. To test the ability of HA35 to promote wound healing in a model of the fetal or neonatal small intestine, we generated 3D intestinal enteroids from NHP ileal tissue and further dissociated this tissue into single cells to create 2D enteroid-derived monolayers (<stro...

Watch this Scientific Journal Video about Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers at JoVE.com

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

In vitro scratch wound assays are commonly used to investigate the mechanisms and characteristics of epithelial healing in a variety of tissue types. ...

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2.2K Views.  University of Oklahoma Health Sciences Center. This protocol describes a method to establish and perform a scratch wound assay on two-dimensional (2D) monolayers derived from three-dimensional (3D) enteroids isolated from non-human primate ileum.

Video: Effect of Hyaluronic Acid 35 kDa on an In Vitro Model of Preterm Small Intestinal Injury and Healing Using Enteroid-Derived Monolayers

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The amount of the different tissues in the callus provides important information on the fracture healing progress. Available in vivo techniques to longitudinally monitor the callus tissue development in preclinical fracture-healing studies using small animals include digital radiography and &#181;CT imaging. However, both techniques are only able to distinguish between mineralized and non-mineralized tissue. Consequently, it is impossible to discriminate cartilage from fibrous tissue. In contrast, magnetic resonance imaging (MRI) visualizes anatomical structures based on their water content and might therefore be able to noninvasively identify soft tissue and cartilage in the fracture callus. Here, we report the use of an MRI-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice. The experiments demonstrated that the fixator and a custom-made mounting device allow repetitive MRI scans, thus enabling longitudinal analysis of fracture-callus tissue development.

In Vivo Evaluation of Fracture Callus Development During Bone Healing in Mice Using an MRI-compatible Osteosynthesis Device for the Mouse Femur

The evaluation of tissue development in the fracture callus during endochondral bone healing is essential to monitor the healing process. Here, we report the use of a magnetic resonance imaging (MRI)-compatible external fixator for the mouse femur to allow MRI scans during bone regeneration in mice.

Endochondral fracture healing is a complex process involving the development of fibrous, cartilaginous, and osseous tissue in the fracture callus. The ...

Intestinal Stem Cell Isolation and Culture in a Porcine Model of Segmental Small Intestinal Ischemia

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ischemia, when the blood supply and therefore oxygen is decreased to the intestine. This leads to intestinal barrier loss and damage to the underlying tissue. Intestinal stem cells reside at the base of the crypts of Lieberk&#252;hn and are responsible for intestinal renewal during homeostasis and following injury. Ex vivo cell culture techniques have allowed for the successful study of epithelial stem cell interactions by establishing culture conditions that support the growth of three-dimensional epithelial organ-like systems (termed "enteroids" and "colonoids" from the small and large intestine, respectively). These enteroids are composed of crypt and villus-like domains and mature to contain all of the cell types found within the epithelium. Historically, murine models have been utilized to study intestinal injury. However, a porcine model offers several advantages including similarity of size as well as gastrointestinal anatomy and physiology to that of humans. By utilizing a porcine model, we establish a protocol in which segmental loops of intestinal ischemia can be created within a single animal, enabling the study of differing time points of ischemic injury and repair in vivo. Additionally, we describe a method to isolate and culture the intestinal stem cells from the ischemic loops of intestine, allowing for the continued study of epithelial repair, modulated by stem cells, ex vivo.

This protocol will enable readers to successfully establish a porcine model of segmental intestinal ischemia and subsequently isolate and culture intestinal stem cells for the study of epithelial repair following injury.

Intestinal ischemia remains a major cause of morbidity and mortality in human and veterinary patients. Many disease processes result in intestinal ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Mongolian Gerbils as an Animal Model of Wound Healing

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving patients' eye health. Historically, corneal healing has been studied in rodents such as mice and rats, but these models might not completely mimic human disorders. However, information on other rodents such as Mongolian gerbils (Meriones unguiculatus) is scant in corneal research.
Here, we describe a technique to develop a novel animal model for studying corneal healing after photorefractive keratectomy. Due to the limited literature available on the cornea of M. unguiculatus, we also describe a histological analysis of the normal cornea. These research techniques can also be employed in the study of eye diseases because of the similarity between the corneas of Mongolian gerbils and humans in terms of genetics, anatomy, and physiology.

This article describes a new animal model developed to study the anatomy and histology of the cornea and its healing processes. This new animal model uses the Mongolian gerbil, which has a cornea with many similarities to the human cornea.

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving ...