The overall goal of this procedure is to induce colitis by infecting mice with the gram-negative mirroring pathogen, citrobacter rodent, allowing the study of pathogen and host interactions in the gastrointestinal tract. This is accomplished by first inoculating, a culture of zero redemption overnight. In the second step, mice are infected by oral gavage with the overnight inoculum.
Next on day seven post-infection, the cecum colon and luminal contents are collected during the final step. The tissues are plated, the bacterial colonies are counted, and the tissues are immunostain for the host proteins of interest. Ultimately, pathogen burden, barrier function, histological damage and changes in host response can be assessed through immunohistochemistry.
Although this method offers a robust model of infectious colitis, allowing for the study of host pathogen interactions and immune responses, it can also be adapted to examine the effects of bacterial infection on the risk of colon cancer. Generally in the individuals new to this method would naturally struggle with the bacterial infection of the mice, with or with, or gage. Begin by using a sterile inoculating loop to streak viable C redemption from a frozen glycerol stock onto an LB electroplate.
After overnight incubation at 37 degrees Celsius, use an inoculating loop to transfer colonies from the LB plate to three milliliters of sterile lb broth. Next, incubate the ERO Dunum culture aerobically in a benchtop incubation shaker at 200 RPM overnight at 37 degrees Celsius. The next morning, the inoculated lb broth should appear cloudy.
Gently agitate the culture to evenly distribute the bacterial cell culture, and then load a one milliliter syringe with broth and attach it to a gastric gavage needle. Select a mouse for gavage by firmly grasping the loose skin over its neck and back with fem and fingers. Then pull back the animal's head with an index finger to immobilize the animal's head and straighten the esophagus for insertion.
Maintaining the mouse in an upright position, direct the bulb tip of the gastric gavage needle along the side of the animal's mouth and over its tongue. Gently pass the needle along the roof of the animal's mouth and advance the needle down the esophagus. Finally, use the syringe to slowly inject 100 microliters of the bacterial inoculum, and then gently remove the gavage needle.
Monitor the breathing rate and behavior of the mouse after returning it, its cage on the day of the assay, scruff the mouse as just demonstrated, and then gavage the animal with 150 microliters of freshly prepared fitzy dextrin. Withdraw food from the cage at this time, four hours after gavage, and then sedation of the animal. Use cardiac puncture to collect as much blood as possible from the anesthetized animal.
Add the blood to a final concentration of 3%acid citrate dextrose in a micro centrifuge tube to deter coagulation. Next, spin down the blood samples for 12 minutes at 1000 cheese and four degrees Celsius. Then collect the serum and dute it to one to 10 and one to 100 in PBS at 100 microliters of each sample.
At these two dilution to an 96 well plate in duplicate. To prepare the standard curve, dilute 100 microliter samples of the original 80 milligrams per milliliter. Fitz Dextrin used to gavage the mice in PBS in dark micro centrifuge tubes.
Then add the standard samples to a 96 well plate in triplicate. Then use a fluorimeter to quantify the fluorescence of each sample and to determine the FXI dextrin concentration using aseptic technique, begin this step by adding one milliliter of sterile PBS and an autoclave metal bead to round bottom to two milliliter centrifuge tubes. Then weigh each tube.
Next, after removing the seum and colon from the mouse, separate the seum from the colon and cut them open longitudinally. Collect the luminal contents with forceps and add the contents to a tube of PBS. Wash the sequel and colonic tissues with sterile PBS before placing them in separate tubes.
Now weigh the tubes of tissue and homogenize the samples using a bead beater for six minutes. At 30 hertz, add 180 microliters of sterile PBS per well to a 96 well plate. Then add 20 microliters of each homogenized sample to the first well in each row and mix well.
Serially dilute the samples by adding 20 microliters from each previous well to obtain dilution from 10 to the minus one in the first well down to 10 to the minus six. Then plate 10 microliters of each dilution from each sample in triplicate on a square bottomed LB plate with a grid after overnight incubation at 37 degrees Celsius. Count the colony forming units on one plate.
Average the triplicates for each sample, and then record the dilution at which each sample was counted. Multiply the average CFU by 50 and by the dilution factor at which the sample was counted, resulting in CFUs per milliliter. Finally, divide by the tissue weight to determine the CFUs per gram after overnight, formal and fixation of the previously saved 0.5 centimeter colon and secum section.
The next morning, wash the tissues with 70%ethanol and place in a cassette to send to a histology facility to be embedded in paraffin and stained with hematin and eosin to de parize the tissues. Place the slides in a coplan staining jar in a 65 degree Celsius water bath for 10 minutes. Now, place the slides in xylene for four washes of two minutes.
Each rehydrate the slides with two five minute washes and 100%ethanol, followed by one five minute wash in each of the following. 95%ethanol, 75%ethanol and distilled water. Place the washed slides in a coplan jar with prewarm sodium citrate buffer, and then place the jar into a steamer for 30 minutes after letting the jar sit at room temperature for another 30 minutes, wash the slides three times with PBS Zied for five minutes.
When the slides are dry, mark the area around the tissue with a pap pen. Then block the tissue with blocking buffer in an immunostaining moisture chamber at room temperature. After one hour, pour off the blocking buffer and incubate the tissues in 50 to 100 microliters of primary antibody at four degrees Celsius overnight.
After the incubation period, wash the tissues three times with PBS Azi for five minutes. Then incubate the slides in 50 to 100 microliters of the secondary antibody in the dark at room temperature after an hour, wash the slides three times with distilled water for five minutes. Finally, dehydrate the tissues, cover the slides with dpi, prolonged gold mounting medium, and then apply a cover slip viewed the slides under a fluorescence microscope.
This first figure shows Fitz dextrin measured in the serum of C 57 black six mice, as well as from toll-like receptor to knockout mice, which have previously been found to exhibit impaired epithelial barrier integrity during infection in the presence of an intact intestinal epithelial barrier. ZI dextrin is poorly permeable through this layer. Therefore, increased levels of Fitz dextrin in serum suggest an impairment of intestinal epithelial barrier integrity during infection, allowing this molecule to leak across as a control serum levels in uninfected mice.
Gavage with Fitz dextrin are also presented. Assent is a luminal pathogen that can intimately adhere to intestinal epithelial cells. Bacterial loads in the luminal contents, sequel and colonic tissues can be at day seven.Post-infection.
Bacterial loads in the colon have been found to peak around 10 to the ninth CFU per gram. Most of the pathology observed during C DUM infection in C 57 PL six mice occurs in the distal two centimeters of the colon macroscopically by day seven post-infection. A thickening of the colonic mucosa, as well as a shortening and length of the colon is observed normally during infection.
C 57 PL six mice suffer only moderate inflammation and pathology as characterized histologically by immune cell infiltration, elongation of colonic crypts and coit cell depletion. To further characterize the host's responses mounted during infection with zero redemption immunofluorescent staining can be utilized to examine changes in the proteins of interest, such as the markers of proliferation, or in examining aspects of the bacterial response such as its localization within the infected tissue. An example of this staining technique, examining the host protein KI 67, a marker for cell proliferation that appears in red and the zero de protein tear, which appears in green to examine bacterial localization at day 12 post infection is illustrated in this figure.
Once mastered, this technique can be performed in minutes if done properly. While attempting this procedure, it's important that the gaging technique be performed properly to ensure that the mice are infected and that the animal's health is monitored throughout the course of the infection. For the last 10 to 15 years, the citrobacter rodent model has become the gold standard for the study of attaching a facing bacterial pathogens such as epec in the heck.
Moreover, it has become a well-recognized model for the study of mucosal immune responses to bacteria, such as the development of TH 17 responses within the gut.
