This method can help answer key questions in the gastroenterology field about the impact of members of the microbiota, specifically those of the genus Helicobacter, on inflammation and cancer. The main advantage of this technique is that it allows the study of Helicobacter infection and its impact on the stomach under physiological conditions. The implications of this technique extend toward the development of therapies for the prevention or elimination of Helicobacter infection or its consequences, including antibiotics, drugs, or vaccines.
These methods provide insight specific to the pathogenesis of Helicobacter infection. However, several of the techniques may be adapted to the study of other gastrointestinal pathogens. Generally, individuals new to this method will struggle with the preparation of a bacterial inoculum capable of establishing an infection in mice.
It is imperative that the Helicobacter isolates are known mouse-colonizing strains that have not undergone extensive subculture in vitro. In addition, the number of in vitro subcultures for these strains should be recorded. Add bacterial suspensions to 15-milliliter polystyrene tubes, and use a plastic loop to transfer one 10-to 20-microliter droplet from each culture onto individual glass microscope slides.
Then, assess the viability and motility of the bacteria under phase-contrast microscopy at a 100 times magnification. Only use the H.pylori inocula if the majority of the bacteria have a bacillary shape. H.felis inocula should primarily contain helical-shaped bacteria.
And load the inocula into individual, disposable, one-milliliter syringes. Equip each syringe with a 23-gauge needle, and use plastic paraffin film to attach a disposable polyethylene catheter to each needle. Next, manually restrain a six-to eight-week-old specific-pathogen-free and Helicobacter-free mouse by the scruff of the neck and tail, and insert one catheter into the center of the open jaw.
Guide the catheter in a caudal toward the esophagus, extending the neck of the mouse to allow ease of access to the stomach through the esophagus and away from the trachea until most or all of the catheter is no longer visible and a resistance is felt. Then, deliver at least one times 10 to the fifth bacteria to ensure an optimal colonization and disease pathology. At the end of the experiment, use fine, curved scissors to open the abdominal cavity for excision of the stomach.
Cut the stomach along the greater curvature, and gently wash the organ two times in one 50-milliliter tube of fresh PBS per wash to remove any residual food. After the second wash, place the tissue into a tared, six-centimeter, plastic Petri dish to record the wet weight. After flattening the stomach, bisect the sample sagittally into two equal tissue fragments comprising the antrum, body, and non-glandular forestomach regions.
Remove the non-glandular region, and weigh one half of each stomach before storing the piece of tissue in the appropriate solution for the planned downstream analysis. Add the other half of stomach tissue to a 15-milliliter conical tube containing 10%formalin, flattening the tissues against the top sides of the tubes after 10 seconds. When the tissues have become affixed to the tube walls, re-immerse the samples in the formalin for at least 24 hours.
To count the number of viable H.pylori in the stomach post-infection, homogenize stomach samples stored in BHI, and perform duplicate serial dilutions of the resulting gastric homogenates in fresh, sterile broth. Divide pre-dried horse blood agar, or HBA, plates supplemented with additional antibiotics into three or four segments, and, using an adaptation of the Miles and Misra technique, add 10 to 100 microliters of each stomach homogenate dilution onto a segment of each agar plate. Spread the homogenates with sterile, plastic loops, and allow the plates to dry.
Then, place the plates in an inverted position in anaerobe gas jars containing a Petri dish of water and a gas pack. Then, incubate the jars at 37 degrees Celsius for four to seven days. When colonies can be observed, enumerate the segments containing between 10 and 100 isolated colonies.
Following euthanasia, the stomach is harvested from the animals and weighed. The non-glandular region is removed, and the stomach is divided into two equal halves comprising the antrum and body. Successful colonization is typically confirmed by performing viable counting and subsequent enumeration of individual colonies from gastric homogenates streaked onto HBA plates.
Note that the presence of contaminating bacteria from the mouse gastric microbiota or large numbers of H.pylori colonies can complicate the enumeration of H.pylori CFUs. Alternatively, PCR can be employed to verify infection using specific validated primers directed at a 325-base pair region of the H.felis and H.pylori ureB genes. In this representative experiment, wild-type C57 Black-6 mice displayed moderate signs of inflammation, including hyperplasia and gland atrophy at six months post-infection with H.felis, as well as cellular infiltration of the submucosa.
More severe inflammation is observed in knockout mice at the same time point, with the additional presence of lymphoid follicles observed in close proximity to the cellular infiltrates. H.felis bacteria can also be visualized in Giemsa-stained sections of infected mouse stomach tissue. Following this procedure, other methods like quantitative PCR, immunohistochemistry, or immunofluorescence can be performed to answer additional questions about the types of host responses induced during Helicobacter infection.
After its development, this technique paved the way for researchers in the field of microbiology and gastroenterology to explore the causative role of Helicobacter pylori in stomach disease in humans. After watching this video, you should have a good understanding of how to reproducibly infect mice with Helicobacter species and to study the pathology associated with the infection. Don't forget that the Helicobacter strains used in this protocol are infectious, and therefore standard biosafety procedures should always be followed while working with these bacteria.
