Enteric glia are becoming increasingly recognized for their roles in intestinal homeostasis and disease processes, including postoperative complications. Equine patients recovering from emergency exploratory laparotomy suffer from a high risk of inflammatory postoperative conditions, highlighting the importance of establishing repeatable equine enteric glial primary cell culture for study.
Inflammatory postoperative conditions of equine colic (acute abdomen) contribute not only to increased client cost, patient discomfort, and 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 µm), yielded a pellet used for plating on 0.05 mg/mL poly-L-lysine-coated wells at a concentration of ~400,000 cells/300 µL of media.
Following confluence and first passage, the enteric glial cells were then exposed to equine recombinant IL-1β (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.
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 1β (IL-1β), 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-1β 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-1β, evaluation of the effect of enteric glial products post IL-1β exposure on the permeability of equine enterocyte monolayers, and possible blockade through the application of equine serum.
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
2. Immunofluorescent cytology
3. Submucosal equine enteric glial primary culture IL-1β exposure
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.
5. Equine enterocyte monolayer permeability following exposure to inflammatory cytokines and equine enteric glial products
6. Statistics
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, Figure 2B). In some instances, microdissection of the submucosal layers was not controlled to a high enough degree, and cultures were contaminated with high percentages of fibroblasts observed through immunofluorescence of fibroblast marker smooth muscle actin (Figure 3).
Next, as an example of the usefulness of equine enteric glial culture, the impact of IL-1β-exposed enteric glia on intestinal epithelial barrier function was assessed. Basolateral exposure to media from equine submucosal enteric glia culture exposed to 10 ng and 25 ng IL-1β for 24 h significantly increased permeability (p = 0.05 and 0.04, respectively, mean of control media = 0.9, mean of glial 10 ng IL-1β group = 0.8, 95% confidence interval = -0.2 to 0.02, mean of glial 25 ng IL-1β group = 0.8, 95% confidence interval = -0.1995 to 0.01060, n = 3-4) (Figure 4A). However, exposure of equine epithelial monolayers to basolateral 10-25 ng equine IL-6, an inflammatory cytokine demonstrated to both be produced by enteric glia exposed to IL-1β and to induce increased enterocyte permeability in rodent models12, did not significantly increase equine enterocyte monolayer permeability (Figure 4B)
Figure 1: Isolation of enteric glia from equine jejunum by microdissection and enzymatic and mechanical digestion. (A) The jejunum was cut along the antimesenteric border and pinned mucosa side up. (B) The intestinal villi were visualized by microscopy and removed with gentle scraping until the wispy lamina propria was visualized. (C) The lamina propria layer was then removed until the smooth muscle fibers were just visible through the translucent submucosa. (D) The submucosa was tented and peeled back from the muscular layer, making cuts to fibrous tags as needed. Images were obtained by a 10x dissection microscope (B,C). Please click here to view a larger version of this figure.
Figure 2: Imaging of the equine submucosal enteric glia cultures. (A) The stellate (arrows), fusiform (open arrowhead) and ganglionic (closed arrowhead) morphology of the equine enteric glia was appreciated by phase contrast microscopy. (B) Purity of cultures was assessed by staining for GFAP, and for the fibroblast marker, smooth muscle actin Scale bars = 100 mm (A), 400 mm (B). Abbreviations: GFAP = glial fibrillar acidic protein; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 3: Imaging of unsuccessful equine submucosal enteric glia cultures. Purity of cultures was assessed by staining for GFAP (red), and for the fibroblast marker, smooth muscle actin (green), demonstrating a large percentage of fibroblast contamination. Yellow arrowhead denotes GFAP and white arrowhead smooth muscle actin. Scale bar = 400 mm. Abbreviation: GFAP = glial fibrillar acidic protein. Please click here to view a larger version of this figure.
Figure 4: Equine epithelial monolayer TEER following inflammatory cytokine and inflammatory glial products exposure. (A) Basolateral exposure to media from equine submucosal enteric glia culture, exposed to 10 ng and 25 ng of IL-1β for 24 h, significantly increased permeability (*p = 0.05 and 0.04, respectively, n = 3-4). (B) Equine enterocyte monolayer acute exposure to basolateral 25 ng of IL-1β and 10-25 ng of equine IL-6 did not significantly increase equine epithelial monolayer permeability (n = 2-4). Please click here to view a larger version of this figure.
Table 1: Reagents for isolation and culture. The following reagents were utilized to formulate media for equine enteric glia isolation, enteric glial culture, and equine intestinal epithelial stem cell culture. Please click here to download this Table.
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 cell population is potentially important because of their role as subepithelial sensors of the intestinal environment, as well as their ability to interact with and communicate with the surrounding cells within the intestinal wall. With their keen sensing ability, it is not difficult to imagine a potentially critical role of glia in equine colic, where intestinal tissue is stressed by distension, ischemia, and potentially surgical manipulation6.
Previous work by Lisowski et al. demonstrated the heightened inflammatory status of equine intestinal tissue at the time of colic surgery. The importance of mitigating this inflammatory environment is highlighted by rodent enteric glia cultures that have proven to be capable of synthesizing products with the ability to disrupt intestinal barrier function7,12. Leakage of intestinal luminal contents into the intestinal wall would not only lead to an inflammatory response but also potential infiltration into the circulation, which could then induce endotoxemic shock/sepsis. Equine colic postoperative patients are unfortunately plagued by numerous inflammatory-based postoperative complications, including sepsis and postoperative ileus, that not only reduce survival and patient comfort but also increase client costs and emotional distress3. Establishing a model of epithelial-glial interactions at the time of colic may elucidate a potential pathway of equine intestinal barrier permeability in colic, and possible prevention.
The culture of equine submucosal enteric glia involves adaptations and limitations due to the characteristics of the species. Most importantly, the length of time required for effective enzymatic digestion required titrating. The authors found that incubation with the enzymes for 2-3 h at 37 °C was necessary for the successful isolation of equine enteric glia. In addition, allowing equine enteric cultures to reach over 80% confluence prior to passaging appears to be detrimental to culture viability. The main limitation of this method at this date is the sample size. This study only involved tissue/cells that had been obtained from horses undergoing colic surgery or euthanasia for non-study-related reasons. Continued culture of equine enteric glia from additional subjects, as well as developing a protocol for freezing and storage of equine enteric glia will greatly increase the power of this study.
This study introduces the novel utility of equine submucosal enteric glial culture and primary culture of equine enterocytes as potential methodologies to help understand equine colic and gastrointestinal disease. In addition, while equine epithelial monolayers grown from isolated intestinal crypts and organoids have been well established in the past, this study not only continued to showcase their usefulness as a model but also encourages additional studies examining the interaction with the equine intestinal enterocyte and other cell types found within the intestinal wall.
Observed interactions between these cell types contain potential applications to both colic and other inflammatory gastrointestinal disorders such as inflammatory bowel disease (IBD) and colitis. In addition, continued refinement of culture methods for enteric glia and enterocytes will allow the establishment of coculture studies in the future. Furthermore, while the current study focuses on the submucosal enteric glial population due to its proximity to the epithelial monolayer and the focus on intestinal barrier function, additional work may be performed to culture and assess the function of the equine enteric glial from the myenteric plexi of the intestinal wall, which may aid in determining the impact of inflammatory equine enteric glia on motility15.
The authors have nothing to disclose.
The authors would like to thank the Morris Animal Foundation for their funding of this project.
1 M HEPES buffer | Gibco | 15630-080 | |
10 mM HEPES | Life Technologies | 15630-106 | |
2 mM GlutaMAX | Life Technologies | 25050-061 | |
4’6-Diaminidino-2-Phenylindol | Invitrogen | D3571 | |
Advanced DMEM/F12 | Life Technologies | 12634-010 | |
Alpha smooth muscle actin antibody | Abcam | 7817 | |
Amphotericin B | Sigma | AA9529 | 4.4 g/mL stock aliquots, final concentration 1.1 µg/mL |
Anti-Antimicotic 1x | Gibco | 15240-096 | |
B27 | Gibco | 12587010 | |
Bovine Serum Albumin | Sigma | AA3311 | |
BSA 50 mg/mL stock solution | Sigma | A3311 | |
CaCL2 | ACROS Organiics | 206791000 | Component of Equine Ringer ‘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’s Stock 2 to make complete “Ringer’s Solution”. |
Collagenase | Sigma | 9891 | |
DMEM-F12 media | Thermo Fisher | 11320033 | |
Donkey anti-rabbit IgG Alexa Fluor 594 | Invitrogen | 21207 | |
EVOM EndOhm dual electrode TEER-measuring chamber | World Precision Instruments | EVM-EL-03-01 | |
EVOM Manual for TEER Measurement | World Precision Instruments | EVM-MT-03-01 | |
G5 | Gibco | 17503012 | |
Gentamicin solution | Sigma | G1272 | Final concentration 20 µg/mL |
GFAP antibody | Abcam | 4674 | |
Goat anti-mouse IgG Alexa Fluor 488 | Invitrogen | 28175 | |
IL-1β ELISA | Thermo Fisher | ESIL1B | |
KCl | Thermo Fisher | P330-500 | Component of Equine Ringer’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’s Stock 2 to make complete “Ringer’s Solution”. |
L-glutamine solution | Corning | 25-00-Cl | |
Matrigel | BD Bioscience | 354277 | |
MgCl2 | Thermo Fisher | M33-500 | Component of Equine Ringer’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’s Stock 2 to make complete “Ringer’s Solution”. |
N2 | Gibco | 17502048 | |
Na2HPO4 | Thermo Fisher | BP332-1 | Component of Equine Ringer’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’s Stock 1 to make complete “Ringer’s Solution”. |
NaCl | Thermo Fisher | S271-10 | Component of Equine Ringer’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’s Stock 2 to make complete “Ringer’s Solution”. |
NaH2PO4 | Thermo Fisher | BP329-500 | Component of Equine Ringer’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’s Stock 1 to make complete “Ringer’s Solution”. |
NaHCO3 | Thermo Fisher | S637-212 | Component of Equine Ringer’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’s Stock 1 to make complete “Ringer’s Solution”. |
Pen/Strep solution | Gemini | 400-109 | |
Poly-L-lysine | Sigma | P2636 | 0.5 mg/mL in 1x borate buffer |
Prism software | GraphPad | ||
Protease | Sigma | P4630 | |
Sodium bicarbonate solution | Sigma | S8761 | 7.5% stock solution |
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