Tissue fibrosis is the primary way that organs fail over time, in response to chronic inflammation or aging. And in fact, I would argue that if you don't die from an infectious agent or cancer, you'll likely die from organ fibrosis as you age. Unfortunately, there're not a lot of great mouse models to mimic human tissue fibrosis.
So what we've developed is a mouse model of Crohn's disease, that's a disease in human that causes fibrosis of the gut and is largely untreatable. In fact, the only way you can treat it is through surgical removal of the fibrotic tissue and re-ligation of the gut. The advantage of our model is that it's fully penetrant in all mouse strains and it very closely mimics human Crohn's disease and the fibrosis persists for over a month, which is really a robust model.
The most challenging part of this procedure is the handling of the mice and especially the oral gavage since that requires a lot of practice special certification. When performing this procedure for the first time, one should be aware that working with Salmonella requires proper safety precautions and sterile technique. Disposal of waste and bacteria should all be planned prior to the experiment.
The visual demonstration of this procedure is very important because it involves the handling of Salmonella that requires special safety precautions as well as proper sterile technique. To begin this procedure, set out a plate with LB agar containing Streptomycin at a concentration of 100 micrograms per milliliter. Using a sterile inoculating loop, streak the plate with a frozen glycerol stock of S typhimurium delta arrow a.
Incubate overnight at 37 degrees Celsius. Streak plates can be stored at four degrees Celsius for up to one week. One day prior to infection, dissolve 0.5 grams of Streptomycin in 2.5 milliliters of water to prepare the antibiotic.
Filter sterilize the Streptomycin solution. Then, use a bulb-tipped 22 gauge gavage needle with a one milliliter syringe to orally gavage the mice with 100 microliters of the streptomycin solution. Next, add three milliliters of LB broth containing Streptomycin at a concentration of 50 micrograms per milliliter to a culture tube.
Using an inoculation loop, inoculate the LB broth with a single colony. Incubate the culture aerobically overnight at 37 degrees Celsius with shaking at 200 rpm. On the day of infection, perform two consecutive one to 10 dilutions of the overnight Salmonella cultures with sterile PBS to prepare the final infection dose.
This results in a 100 microliter inoculum containing approximately three million colony forming units. After this, use a bulb-tipped 22 gauge gavage needle with a milliliter syringe to gavage each mouse with 100 microliters of the prepared Salmonella. First, set out two milliliter safe-lock round bottom microtubes, add one milliliter of sterile PBS and an autoclaved stainless steel bead to each tube.
Pre-weigh the tubes prior to tissue collection. After euthanizing mice, resect their cecal and splenic tissues, making sure to collect tissue from individual animals into separate tubes. Weigh each tube to determine the tissue weights.
Use a mixer mill apparatus to homogenize the tissues at 30 hertz for 15 minutes. Then, transfer 900 microliters of PBS into each well of a 96-well two milliliter mega block. Pipette 100 microliters of tissue homogenates into the first well, and mix well.
Perform serial dilutions by adding 100 microliters to subsequent wells, until a 10 to the negative sixth dilution is obtained. Plate 10 microliters of each dilution in triplicates onto LB agar that contains Streptomycin. Then, count the average colony forming units and determine the colony forming units per gram of tissue as outlined in the text protocol.
Open Fiji, which is an open sourced imaging software and drag and drop the tif image file onto the tool bar. On the menu bar, select Image, Type, RGB stack to split the image into red, green and blue channels. Slide the horizontal bar at the bottom of the panel to set the channel to green.
Open Image, Adjust, Threshold tool. Adjust minimum and maximum limits to eliminate any background signals. Once the desired threshold is set, close the threshold tool and go to Analyze, Set Measurements, check off Area, Area fraction, Limit to threshold and Display label.
Get the tissue selection with either the freehand selections or polygon selections tool and measure the percent area positive for collagen by clicking Analyze, Measure. Then, normalize the absolute area positive for collagen staining to tissue area. Streptomycin treatment followed by oral infection with S typhimurium delta arrow a leads to robust intestinal inflammation and fibrosis, especially in the cecum.
Typical pathogen burdens of 100 million to one billion colony forming units can be recovered per gram of cecum from infected animals, while 10 thousand colony forming units can typically be recovered per gram of spleen. Assessment of fibrosis in picrosirius red stained cecal sections indicates peak fibrosis 21 days after infection, while much of the pathology is resolved by day 42 post infection. Thorough preparation of materials is essential as this experiment requires multiple days to set up.
Preparation of LB plates and Salmonella and Streptomycin should all be done aseptically. Prior to the experiment, ensure that the experimenter is familiar with the animal protocol as well as safety hazards. Upon assessing the collagen burden, the experimenter may choose to perform other biological assays, such as RNA sequencing to assess gene expression profiles or flow cytometry to assess immune cell subsets or a tissue ELISA to assess cytokine profiles.
So our model is particularly good at mimicking a human disease called Crohn's disease where you get fibrosis of the gut, which is largely untreatable and the only way you can treat it is to remove a section of the gut and stitch it back together and hope it doesn't come back. Our model's very robust and we've already shown that targeting innate lymphoid cells type three, a transcription factor called raw alpha, or interleukin 17 ameliorates the disease and prevents the fibrosis. Our model is particularly good at identifying the factors that play in the pathogenesis of Crohn's disease and gut fibrosis and we're already on the road to identifying a number of targets that could be treated therapeutically to alleviate inflammatory bowel disease.
Our model has already entered the same ILC3 or alpha NIL17 access may be at play in other fibrotic disease and these include things like psoriasis, multiple sclerosis, idiopathic pulmonary fibrosis. So it's possible that targeting these same factors might alleviate fibrosis in those diseases as well. Although the mutant Salmonella stream isn't virulent, it is important to follow the standard biohazard safety protocols given by the facility.
Also, since formaldehyde vapors are known to be carcinogenic, it's important to work behind a fume hood during the fixation step.
