Name: Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut (Video) | JoVE Journal
Uploaded: 2011-05-24T12:00:00.000+00:00
Duration: 10 min 5 s
Description: Learn to establish a Trichuris muris infection model in mice for studying Th2 immunity and intestinal inflammation. This method enables analysis of resistant versus susceptible immune responses using ELISA and histology.

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5 minutes

This work was supported by the Canadian Institutes of Health Research (MSH-95368, MOP-89773 and MOP-106623 to C.Z.) and the Canada Foundation for Innovation. S.C.M. is the recipient of a CIHR/Canadian Association of Gastroenterology postdoctoral fellowship. C.Z. is a CIHR New Investigator.

1. Propagating Trichuris muris eggs
<ol>
<li>To generate new batches of Trichuris muris eggs, infect 20-30 immunodeficient mice (eg. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ 
(NSG) or 129S6/SvEvTac-Rag2tm1Fwa (RAG2-/-)) or genetically susceptible mice (eg. AKR/J) that are 6-8 weeks old with approximately 300 Trichuris 
muris eggs by oral gavage.</li> 
<li>After 32–35 days sacrifice the mice by CO2 asphyxiation.</li>
<li>Expose the ventral side of the mouse and wet the abdomen with 70% ethanol.</li>
<li>Using forceps grasp the abdominal skin and make a small incision using scissors. Cut the skin to expose the abdominal contents.</li>
<li>Identify the cecum and the proximal large intestine, and remove them.</li>
<li>Place the cecum and proximal large intestine in a 10 cm petri dish containing Dulbecco's Modified Eagle Medium (DMEM) with 500 U/ml penicillin and 500 μg/ml 
streptomycin (P/S). Cut the cecum open to expose the lumen and the worms. Using forceps place cecum in a beaker of phosphate buffered saline (PBS) and shake gently to remove feces.</li>
<li>Return cecum to petri dish and using smooth curved forceps (Roboz RS-5047) gently pull out worms and place them in a well of a 6 well plate containing 5 mls of DMEM + P/S.</li>
<li>Repeat this process for all of the mice.</li>
<li>Put 6 well plate in a Tupperware container with a damp paper towel to maintain humidity and incubate at 37°C for 4 hrs.</li>
<li>After 4 hrs remove worms using smooth curved forceps and split them into 2 new wells of the 6 well plate containing 5mL DMEM + P/S. Harvest the contents of the original well and place in a 15 ml falcon tube. Spin tube at 3000 rpm and remove and reserve supernatant as 4 hr antigen (can be used for cell restimulation after PBS dialysis, where typical protein yields are 10-20 mg per 20 mice) and resuspend egg-containing pellet in autoclaved tap water.</li>
<li>Put the 6 well plate back at 37°C overnight. Remove and dispose of the worms. Harvest the contents of the wells into a falcon tube and spin at 3000 rpm for 5 min. Remove supernatant and reserve as overnight antigen (can be used for Trichuris Ag specific antibody isotype ELISAs after PBS dialysis, where typical protein yields are 10-20 mg per 20 mice) and resuspend egg-containing pellet in autoclaved tap water.</li>
<li>Combine 4 hr and overnight eggs and filter the eggs through the 70μm strainer to remove any leftover worms then transfer into a 75 cm2 flask. Keep 
flask in a dark place covered in foil for 6 weeks at room temperature (RT).</li>
<li>Wash eggs in autoclaved tap water and resuspend at 50 viable eggs in 50 μl. To do this, centrifuge eggs at 3000 rpm for 5 min and resuspend in ˜50ml sterile tap water. Remove 50 μl eggs and place on a glass slide. Using the 40x magnification count the number of viable embryonated eggs in the 50 μl. Dilute eggs to give 50 viable eggs in 50 μl. For images of viable eggs please see the study by Kopper and Mansfield16.</li>
</ol>
2. Acute infection of experimental mice with Trichuris muris eggs (200 eggs/mouse)
<ol>
<li>Thoroughly resuspend eggs and remove enough volume to treat the number of mice X 2 (eg. for 10 mice take 200 μl x 10 mice x 2 = 4 mls of eggs) and place them 
in a 14 ml snap cap tube.</li>
<li>Invert the 14 ml snap cap tube to mix. Bring up 200 μl of eggs into 1 ml syringe on an appropriate size feeding needle (for mice weighing 25 grams we used 18 gauge 1 1/2" needles). In one hand scruff the mouse and 
using the other hand administer eggs by oral gavage .</li>
<li>Wait 21 days.</li> 
</ol>
3. Experimental harvest
<ol>
<li>On day 21 sacrifice the mice using CO2.</li>
<li>Collect blood from mice by cardiac puncture, in a non-heparinized syringe, and store at 4°C. The following day isolate serum and freeze at -20°C. Serum can be used for detection of Trichuris-specific IgGs and IgE by ELISA.</li>
<li>Expose the ventral side of the mouse and wet the abdomen with 70% ethanol.</li>
<li>Using forceps grasp the abdominal skin and make a small incision using scissors. Cut the skin and underlying membrane to expose the abdominal contents.</li> 
<li>Identify the small intestine, cecum and colon. Grab the cecum with forceps and pull out of abdominal cavity. With another pair of forceps push small intestine over to the right exposing the mesenteric lymph nodes (mLNs). Remove the mLNs being careful not to cut into the intestines and place in a 15 ml falcon containing 2 mls of media. If desired these can be processed and restimulated in vitro (4x106/ml in 1 ml in 24 well plates with 1 μg/ml αCD3/CD28 or with 50μg/ml 4h Trichuris Ag) for 72 hrs for cytokine analysis by ELISA and intracellular flow cytometry.</li>
<li>Cut out the cecum and colon.</li> 
<li>Using forceps, push out fecal pellets from distal colon and place in a 2 ml Axygen tube. Freeze at -20°C. Fecal pellets can be analyzed by western blot for expression of resistin-like molecule-β (RELM-β).</li>
<li>Remove the tip of the cecum (˜0.5 cm). To remove feces hold cecum tip with forceps and flush with PBS using a 3 ml syringe and 22-gauge needle in a petri dish. Place in fresh 4% paraformaldehyde in a 5 ml Falcon snap cap tube. Store at 4°C. These can be paraffin-embedded, sectioned and mounted on slides for histological analysis.</li>
<li>Remove a piece of proximal colon (˜ 1 cm) for RNA. Put sample into 2 ml Axygen tube with 500 μl RNAlater. Store at 4°C. These samples can be analyzed for mRNA expression by qPCR.</li> 
<li>Place the rest of the cecum in a labeled petri dish and freeze at -20°C.</li>
</ol>
4. Enumeration of Trichuris muris worms in the cecum
<ol>
<li>Remove cecum from freezer and place in a petri dish containing ˜5 ml water.</li>
<li>Using scissors, cut open cecum to expose the lumen.</li>
<li>Hold the cecum with forceps and vigorously shake the cecum to remove the majority of the feces.</li>
<li>Transfer cecum to another petri dish containing water and using smooth curved forceps gently scrape off the IECs the underlying tissue.</li>
<li>Transfer cecum to a new petri dish containing water and vigorously scrape remaining tissue with forceps, ripping the tissue into small pieces.</li> 
<li>Use a dissecting microscope to count all of the worms in all 3 of the petri dishes. As you find a worm remove it from the dish to ensure the same worm isn’t counted twice. If worms are damaged (i.e. broken in half) count only thick or thin ends to get an accurate count.</li>
</ol>
5. Representative Results
The Trichuris muris infection model allows researchers to quantify parasite burden and systemic immune responses, as well as to histologically examine the inflammatory response within the distal cecum. When the lumen of the harvested cecum is exposed, Trichuris worms can be enumerated using a dissecting microscope (Figure 1A-B). Parameters of immunity to Trichuris also include antibody isotype switching to IgG1 (associated with Th2-type immunity) in resistant animals, and IgG2a (associated with Th1-type immunity) in susceptible animals (Figure 1C). Expression of the goblet cell-specific protein resistin-like molecule-β (RELM-β) is associated with clearance of Trichuris and is detectable by Western blot analysis (Figure 2). The distal portion of the cecum can be stained with Periodic Acid-Schiff (PAS) to assess goblet cell responses and the extent of intestinal inflammation in response to infection (Figure 3).
<img src="/files/ftp_upload/2774/2774fig1.jpg" alt="Worm burden comparison, graph data analysis, microscopy images of nematodes, dilution experiment." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Quantification of immunity to Trichuris 
muris. (A) Resistant (B6) mice expel Trichuris muris by day 21 post-infection. By contrast, susceptible (AKR/J) mice become chronically infected 
with Trichuris, resulting in parasite establishment. Each bar represents the mean ± SEM of 4-8 animals per group. (B) Worms are counted using a 
dissecting microscope. Top panel shows worms in isolation, bottom panel shows worms in the presence of IECs and feces. (C) Trichuris-specific IgG2a 
titres are determined by ELISA of diluted serum (1:2 dilution, starting at 1:20 dilution and ending at 1:2560) on plates coated with excretory Trichuris 
antigen (O/N Ag, 5 μg/ml). Susceptible AKR/J mice have notably higher levels of Trichuris-specific serum IgG2a than resistant B6 mice.
<img src="/files/ftp_upload/2774/2774fig2.jpg" alt="Western blot; protein expression comparison; B6 vs AKR; N vs Tm treatment; molecular biology." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Clearance of Trichuris muris is associated with expression of the goblet cell-specific protein RELM-β7. 
RELM-β production can be detected from fecal pellets from Trichuris-infected resistant (B6), but not susceptible (AKR/J) mice by western blot 
analysis. Each lane is representative of a single animal, (N)=Naive, (Tm)=21-day Trichuris infected mice.
<img src="/files/ftp_upload/2774/2774fig3.jpg" alt="Histological analysis of B6 and AKR mouse colon; naive and Trichuris infection; microscope images." data-altupdatedat="1788235205000" data-alt="Figure 3"> Figure 3. Histological examination of the distal cecum 
following Trichuris infection. 5-7 μm sections of the distal cecum stained with PAS. Goblet cell hyperplasia and mucus production are 
evident in resistant (B6) but not susceptible (AKR/J) mice following Trichuris infection.

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<li>Else, K. J., Finkelman, F. D., Maliszewski, C. R., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine-mediated+regulation+of+chronic+intestinal+helminth+infection%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">Cytokine-mediated regulation of chronic intestinal helminth infection.</a> J. Exp. Med. 179, 347-351 (1994).</li>
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<li>Bancroft, A. J., McKenzie, A. N., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+critical+role+for+IL-13+in+resistance+to+intestinal+nematode+infection%5BTitle%5D)+AND+%22J.+Immunol%22%5BJournal%5D)">A critical role for IL-13 in resistance to intestinal nematode infection.</a> J. Immunol. 160, 3453-3461 (1998).</li>
<li>Helmby, H., Takeda, K., Akira, S., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin+%28IL%29-18+promotes+the+development+of+chronic+gastrointestinal+helminth+infection+by+downregulating+IL-13%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">Interleukin (IL)-18 promotes the development of chronic gastrointestinal helminth infection by downregulating IL-13.</a> J. Exp. Med. 194, 355-364 (2001).</li>
<li>Owyang, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interleukin+25+regulates+type+2+cytokine-dependent+immunity+and+limits+chronic+inflammation+in+the+gastrointestinal+tract%5BTitle%5D)+AND+%22J.+Exp.+Med%22%5BJournal%5D)">Interleukin 25 regulates type 2 cytokine-dependent immunity and limits chronic inflammation in the gastrointestinal tract.</a> J. Exp. Med. 203, 843-849 (2006).</li>
<li>Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RELM%CE%B2%2FFIZZ2+is+a+goblet+cell-specific+immune-effector+molecule+in+the+gastrointestinal+tract%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci.+U.+S.+A%22%5BJournal%5D)">RELMβ/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract.</a> Proc. Natl. Acad. Sci. U. S. A. 101, 13596-13600 (2004).</li>
<li>Datta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+novel+genes+in+intestinal+tissue+that+are+regulated+after+infection+with+an+intestinal+nematode+parasite%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Identification of novel genes in intestinal tissue that are regulated after infection with an intestinal nematode parasite.</a> Infect. Immun. 73, 4025-4033 (2005).</li>
<li>Cliffe, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accelerated+intestinal+epithelial+cell+turnover%3A+a+new+mechanism+of+parasite+expulsion%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Accelerated intestinal epithelial cell turnover: a new mechanism of parasite expulsion.</a> Science. 308, 1463-1465 (2005).</li>
<li>Artis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+weapons+in+the+war+on+worms%3A+identification+of+putative+mechanisms+of+immune-mediated+expulsion+of+gastrointestinal+nematodes%5BTitle%5D)+AND+%22Int.+J.+Parasitol%22%5BJournal%5D)">New weapons in the war on worms: identification of putative mechanisms of immune-mediated expulsion of gastrointestinal nematodes.</a> Int. J. Parasitol. 36, 723-733 (2006).</li>
<li>Finkelman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytokine+regulation+of+host+defense+against+parasitic+gastrointestinal+nematodes%3A+lessons+from+studies+with+rodent+models%5BTitle%5D)+AND+%22Annu.+Rev.+Immunol%22%5BJournal%5D)">Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models.</a> Annu. Rev. Immunol. 15, 505-533 (1997).</li>
<li>Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enteric+helminth+infection%3A+immunopathology+and+resistance+during+intestinal+nematode+infection%5BTitle%5D)+AND+%22Chem.+Immunol%22%5BJournal%5D)">Enteric helminth infection: immunopathology and resistance during intestinal nematode infection.</a> Chem. Immunol. 66, 41-61 (1997).</li>
<li>Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+intestinal+muscle+contraction+by+interleukin-9+%28IL-9%29+or+IL-9+neutralization%3A+correlation+with+worm+expulsion+in+murine+nematode+infections%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">Modulation of intestinal muscle contraction by interleukin-9 (IL-9) or IL-9 neutralization: correlation with worm expulsion in murine nematode infections.</a> Infect. Immun. 71, 2430-2438 (2003).</li>
<li>Khan, W. I., Blennerhasset, P., Ma, C., Matthaei, K. I., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stat6+dependent+goblet+cell+hyperplasia+during+intestinal+nematode+infection%5BTitle%5D)+AND+%22Parasite+Immunol%22%5BJournal%5D)">Stat6 dependent goblet cell hyperplasia during intestinal nematode infection.</a> Parasite Immunol. 23, 39-42 (2001).</li>
<li>Akiho, H., Blennerhassett, P., Deng, Y., Collins, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+IL-4%2C+IL-13%2C+and+STAT6+in+inflammation-induced+hypercontractility+of+murine+smooth+muscle+cells%5BTitle%5D)+AND+%22Am.+J.+Physiol.+Gastrointest.+Liver+Physiol%22%5BJournal%5D)">Role of IL-4, IL-13, and STAT6 in inflammation-induced hypercontractility of murine smooth muscle cells.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 282, 226-2232 (2002).</li>
<li>Kopper, J. J., Mansfield, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+improved+methods+for+delivery+of+Trichuris+muris+to+the+laboratory+mouse%5BTitle%5D)+AND+%22Parasitol.+Res%22%5BJournal%5D)">Development of improved methods for delivery of Trichuris muris to the laboratory mouse.</a> Parasitol. Res. 107, 1103-1113 (2010).</li>
<li>Bancroft, A. J., Else, K. J., Grencis, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-level+infection+with+Trichuris+muris+significantly+affects+the+polarization+of+the+CD4+response%5BTitle%5D)+AND+%22Eur.+J.+Immunol%22%5BJournal%5D)">Low-level infection with Trichuris muris significantly affects the polarization of the CD4 response.</a> Eur. J. Immunol. 24, 3113-3118 (1994).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by The University of British Columbia.

This protocol details a standard high dose acute Trichuris muris infection which can be modified as required by the investigator. For example, mice can be sacrificed and tissues harvested on different days. To determine that the mice have successfully established full worm burden they can be sacrificed on day 14, at which point all mice should carry a burden of approximately 200 worms. Mice can also be infected for 32 days where any worm detected will have reached maturity and would remain with host for the d...

<table><tbody><tr><td>Animal Feeding Needles (18 x 1&amp;frac12;&quot;)</td><td>Popper &amp;amp; Sons, Inc.</td><td>7912</td><td></td></tr><tr><td>Smooth curved Forceps</td><td>Roboz Surgical Instruments Co.</td><td>RS-5047</td><td></td></tr><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td>11965</td><td></td></tr><tr><td>NOD.Cg-&lt;em&gt;Prkdc&lt;sup&gt;scid&lt;/sup&gt; Il2r&lt;sup&gt;gtm1Wjl&lt;/sup&gt;/SzJ&lt;/em&gt; (NSG)</td><td>Jackson Laboratory</td><td>005557</td><td>These are the mice we used, however, any immunodeficient mice or susceptible strain should work.</td></tr><tr><td>RNAlater</td><td>Qiagen</td><td>76104</td><td></td></tr><tr><td>2 ml tubes</td><td>Axygen Scientific</td><td>MCT-200-C</td><td></td></tr><tr><td>15 ml tubes</td><td>Falcon BD</td><td>352096</td><td></td></tr><tr><td>6 well plates</td><td>Falcon BD</td><td>353046</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>&amp;#945;-RELM&amp;#946; antibody</td><td>PeproTech Inc</td><td>0694270Rb</td><td></td></tr></tbody></table>

trichuris muris infection: a model of type 2 immunity and inflammation in the gut

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

The overall goal of this procedure is to establish and maintain a natural hain infection model. First mice are infected with tra curus mirrors eggs by oral gavage. 21 days later, the infected animals are euthanized and blood mesenteric, lymph nodes, fecal pellets, colons, and seeker are harvested.Using these samples, the immune response generated against tricks. Mirrors can be analyzed. Serum samples can be analyzed by lysa to detect TRA specific antibodies.In vitro restimulation of lymph node cells can be performed to analyze cellular immune responses. Western blotter fecal pellets can be used for protein content assessment and gene expression analysis can be performed by QPCR analysis of the colon. Finally, the cecum tip can be used for histology while the rest of the cecum may be used to determine the worm burden in infected mice.Ultimately, results can be obtained that show if a mouse is resistant or susceptible to trais MOUs infection and the immune response mounted against the parasite can be characterized. This method can help answer key questions in the immunology field, such as which factors control T helper cell activation, as well as which proteins are required for TH two cell differentiation. Trauss MOUs eggs can be propagated in immunodeficient mice or genetically susceptible strains such as a KR 35 days after infection to harvest eggs from euthanized mice.Expose the animal's ventral surface and rinse the mouse's abdomen with ethanol using sterile forceps and scissors. Grasp the abdominal skin and make a small incision. Expose the abdominal contents, identify the cecum and the proximal colon, and then remove them.Transfer the organs into a 10 centimeter Petri dish containing penicillin and streptomycin, supplemented DMEM. Then cut the cecum and proximal colon open, exposing the lumen and worms using forceps. Place the open organs in a beaker containing PBS and gently shake to wash off feces.Once the cecum has been washed, transfer it to a fresh Petri dish and use smooth curved forceps to gently pull out the worms. Transfer all the worms from each mouse into one well of a six Well plate containing five milliliters of antibiotic supplemented DMEM. Place the six well plate with a damp paper towel inside a Tupperware container and incubate at 37 degrees Celsius for four hours to allow the worms to lay eggs.Following this incubation, use forceps to remove the worms from the original well and split them into two new wells containing DMEM. Splitting the worms in this manner will maximize egg recovery using a pipette. Harvest the remaining medium containing secreted antigen and eggs from the original well and transfer it to a 15 milliliter Falcon tube.Centrifuge the tube at 3000 RPM for five minutes. Then remove and save the supinate containing four hour antigen and Reese's bend the egg containing pellet in sterile water. Place the six well plate containing worms at 37 degrees Celsius once more and incubate it overnight the next morning.After discarding the worms, harvest the contents of the wells as shown earlier and centrifuge the supernatant to isolate the eggs. Save the supernatant containing overnight antigen and resuspend the eggs in water. As before, combine these eggs with the eggs obtained from the four hour incubation.Filter the eggs through a 70 micron strainer to remove any leftover worms. Then transfer them into a 75 centimeter square flask. Cover the flask in aluminum foil to shield it from light and store the eggs for six weeks at room temperature.This will allow the eggs to mature and embry anate after six weeks. Wash the eggs and stir our water and count viable eggs. Seen here.As an example of a viable embryonated egg and a non-viable egg, Reese's bend the Exeter concentration of 50 per 50 microliters. At this point, the eggs may be stored at four degrees Celsius before infecting mice. Be sure to thoroughly resend the egg containing solution.Transfer the desired amount of inoculum into a 14 milliliter snap cap tube and invert the tube to mix. Draw up 200 microliters of eggs into an appropriate size feeding needle for animal size fitted to a one milli liter syringe. Scruff the mouse and administer the eggs by oral gavage.Return the infected mouse to its cage and allow the infection to proceed for 21 days. After 21 days, use a non heparinized syringe to harvest the blood of infected mice by cardiac puncture. Following carbon dioxide euthanasia, the blood samples can be stored at four degrees Celsius.For serum isolation and analysis of tra specific IgGs and IgE by Eliza. Expose the mouse's abdominal cavity as described earlier. Then use forceps to pull out the cecum with scissors or another pair of forceps.Push the small intestine over to the right, exposing the mesenteric lymph nodes. Gently remove the lymph nodes while being careful not to cut into the intestines and place them into a 15 milliliter Falcon tube containing two milliliters of media. Lymph node cells can later be reed in vitro with four hour antigen S supernatant from the worm cultures to analyze the cellular immune response to the parasite.Once the lymph nodes have been harvested, excise the secum and colon as described earlier, using forceps, liberate fecal pellets from the distal colon, placing them in the two milliliter oxygen tube pellets can be stored at minus 20 degrees Celsius and analyzed for protein content by western blot using scissors. Cut approximately one centimeter of proximal colon and place in a tube containing RNA. Later store at four degrees Celsius.Cut off about five millimeters from the tip of the cecum holding the tip of the cecum with forceps. Use a three milliliter syringe and a 22 gauge needle to flush the sample with PBS. Then transfer it to a five milliliter flk and snap cap tube containing 4%paraldehyde.The paraldehyde fixed cecum sample can be stored at four degrees Celsius and used for histological analysis. Place the rest of the cecum in a Petri dish and freeze it at minus 20 degrees Celsius to kill worms. For easier enumeration to enumerate worms, thaw the secum and place it in a Petri dish containing about five milliliters of water using scissors.Cut the secum open to expose the lumen and shake to collect feces into a dish. Place the clean secum into a fresh Petri dish containing water and use smooth curved forceps to scrape off the intestinal epithelial cells. Transfer the secum to a new Petri dish containing water and rip the tissue up into small pieces.E observe the feces, intestinal epithelial cells, and cecum tissue under a dissecting microscope and enumerate the worms, removing them as they're counted and placing them in a fresh dish. This is how the worms in the cecum should look before and after isolation as shown here. Following tragus mirrors infection black six are able to control the parasites while a KR mice develop elevated parasite burdens.Similarly susceptible A KR mice develop markedly higher TRA specific IgG two a titers than black six mice as determined by serum Eliza. In this experiment, Western blot analysis revealed that the amount of goblet cell specific protein realm beta was increased in the feces of black six, but not a KR mice following infection. Histological analysis of the distal cecum shows goblet cell hyperplasia and mucus production in resistant TRAs MOUs infective black six mice, but not in susceptible a KR mice.After watching this video, you should have a good understanding of how to propagate trismus eggs, infect experimental mice, and harvest the intestinal tissues, meso, enteric lymph nodes, and blood for analysis of infection in susceptible or resistant mice.

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Research

JoVE Journal

Immunology and Infection

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis.  Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

Citrobacter rodentium infection provides a valuable model to study enteric bacterial infections as well as host immune responses and colitis in mice. This protocol outlines the measurement of barrier integrity, pathogen load and histological damage allowing for the thorough characterization of pathogen and host contributions to murine infectious colitis.

The Citrobacter rodentium Mouse Model: Studying Pathogen and Host Contributions to Infectious Colitis

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

In this antigen-driven colitis model, OT-II CD4+ T cells expressing a red fluorescent protein were adoptively transferred into RAG-/- mice that express a green fluorescent protein in mononuclear phagocytes (MPs). The hosts were challenged with Escherichia coli (E.coli) expressing the ovalbumin protein (OVA) fused to a cyan fluorescent protein (CFP).

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells

Here a protocol to mimic the entrance of bacterial-derived compounds after intestinal barrier breach is presented. A low sublethal dose of lipopolysaccharide was injected systemically into mice, which were monitored for 24 h post-injection. The expression of pro-inflammatory cytokines was determined at several time points in spleen, liver, and colon.

Injections of Lipopolysaccharide into Mice to Mimic Entrance of Microbial-derived Products After Intestinal Barrier Breach

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

This protocol describes a mouse model of Salmonella driven intestinal fibrosis that resembles key pathological hallmarks of Crohn’s disease including transmural inflammation and fibrosis. This method can be used to evaluate host factors that alter fibrotic outcomes using mutant mice maintained on a C57Bl/6 genetic background.

Chronic Salmonella Infection Induced Intestinal Fibrosis

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

Learn to establish a Trichuris muris infection model in mice for studying Th2 immunity and intestinal inflammation. This method enables analysis of resistant versus susceptible immune responses using ELISA and histology.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

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