The overall goal of this experiment is to present a model that mimics the entrance of microbial-derived products after an intestinal barrier breach. This model can be used to investigate immune responses after invasion of microorganisms. This method can help answer key questions in the inflammatory bowel disease field, such as uncontrolled inflammation, which is characterized by an increased intestinal epithelial permeability.
The main advantage of this technique is that it not only uses a defined pump, but is also an uncomplicated approach that does not require surgery and can be used in every laboratory setting. Demonstrating the procedure will be myself, Rachel Mak'Anyengo, a postdoc, and Berna Kaya, a PhD student. Handle the mouse gently but firmly, and restrain the animal in one hand.
Ensure that the mouse is securely held and can breathe normally. Tilt the mouse nose slightly towards the floor to expose the abdomen for injection. Locate the midline of the abdomen and inject the volume of LPS required on the lower-left or right side.
It is very important to ensure that the needle enters the peritoneal cavity to avoid misinjections. The needle should also not go too deep into the peritoneal cavity to avoid injury of inner organs. Gently return the animal to the home cage.
Monitor the mice for the occurrence and severity of endotoxemia at the time of injection and every two hours thereafter for eight hours. Record observations in the attached score sheet. Prepare the collection tubes by adding one milliliter of single-step RNA isolation reagent to two milliliter tubes pre-filled with 1.4 millimeter ceramic spheres.
After euthanizing and ensanguinating the mouse, use a pair of scissors to cut the skin and the muscle layer, exposing the abdominal cavity with the inner organs. Carefully dissect the spleen, liver, and colon. Clean the fat from each organ, then place in PBS on ice.
Next, use scissors or a scalpel to cut a 0.5 centimeter long piece from each organ. Briefly dry the tissue on a piece of paper tissue, and place it in the collection tube, making sure it is completely immersed in RNA isolation reagent. Then homogenize the tissue in a high-speed benchtop homogenizer.
For soft tissues like spleen, liver and colon, use one cycle of homogenization at high speed for 30 seconds. After homogenization, incubate the samples for five minutes at room temperature. Carefully immerse the tubes in liquid nitrogen to snap freeze the tissue lysate.
Store the snap-frozen lysate at minus 80 degrees Celsius until RNA isolation. Begin RNA isolation by thawing the frozen tissue lysate on ice. Once thawed, centrifuge the sample at 1000 times G for five minutes at four degrees Celsius to pellet the remaining tissue particles that might be left.
Following centrifugation, transfer the supernatants to a new, nuclease-free 1.5 milliliter tube, and add 200 microliters of ice-cold chloroform per one milliliter of RNA isolation reagent. Immediately shake the gristly for 10 to 15 seconds. After incubating the samples for two to three minutes at room temperature, centrifuge at 12, 000 times G for 15 minutes at four degrees Celsius.
The sample will now contain three phases, the lower red phenol chloroform phase, the interphase, and the upper colorless aqueous phase. Collect the RNA-containing upper aqueous phase and transfer into a new, nuclease-free 1.5 milliliter tube. To precipitate the RNA, add 0.5 milliliters of ice-cold isopropanol per one milliliter of RNA isolation reagent, and mix gently by inverting the tube five to six times.
After incubating for 10 to 15 minutes at room temperature, centrifuge at 12, 000 times G for 10 minutes at four degrees Celsius. Following centrifugation, completely remove the supernatant containing the isopropanol without disturbing the pellet. Then wash the pellets with one milliliter of ice-cold 75%ethanol per one milliliter of RNA isolation reagent used for the initial lysis, and vortex the sample briefly.
After centrifuging the sample at 7500 or 12, 000 times G for five minutes at four degrees Celsius, completely remove the supernatant without disturbing the pellet. Leave the opened tubes under the chemical hood for three to five minutes to air-dry the RNA pellet at room temperature. Next, dissolve the RNA pellet in 20 to 50 microliters of nuclease-free water by pipetting carefully up and down.
Then incubate the sample at 55 degrees Celsius for 10 to 15 minutes to further improve the solution of the RNA. Digest contaminating DNA by first adding nuclease-free water to a maximum of 10 micrograms of RNA so that the final volume will be 50 microliters after the addition of buffer and enzyme. Then add five microliters of 10X DNase 1 buffer and one microliter of recombinant DNase 1 per sample and mix gently by pipetting up and down.
Incubate at 37 degrees Celsius for 20 to 30 minutes. Following the incubation, vortex the DNase in activation reagent, then add five microliters to the sample and mix well. Incubate for five minutes at room temperature, mixing occasionally by hand.
After centrifuging at 10, 000 times G for one and a half minutes, transfer the supernatant containing the DNA-free RNA into a new, nuclease-free tube. Finally, measure the RNA concentration and quality by spectrophotometer. After injection of LPS, the disease score, which includes assessments of the appearance and activity of the animals, opening of their eyes, and their respiration rate and quality, was plotted on the Y axis against time on the X axis.
QPCR to quantify cytokine expression revealed that Il6 peaked two hours after LPS injection in spleen and colon, and four hours after injection in the liver. The expression of Il1 beta, Tnf alpha, and Il10 peaked four hours after injection in all tissues. Within eight hours, the expression of Il6, Il1 beta, Tnf alpha and Il10 returned to baseline levels.
While attempting the procedure, it's important to remember to consider the dose of LPS, the hygiene status of the mice, the time point of the experiment termination and most importantly, the genetic background of the mouse strain. After watching this video, you should have a good understanding of how to mimic the entrance of bacterial-derived compounds into a host after a barrier breach. The low, sublethal dose of LPS systematically injected into mice induces upregulation of proinflammatory cytokines, which are quantified by RGQ PCR analysis.
