This protocol describes the induction of Gram-negative monobacterial sepsis in a mouse model system. The model is useful in investigating the inflammatory and lethal host responses during sepsis.
Sepsis is a dysregulated host immune response to microbial invasion or tissue damage, leading to organ injury at a site distant from that of infection or damage. The general public has become more aware of this disorder, during the COVID-19 pandemic. In 2017, there were 48.9 million sepsis incidences, and 11 million deaths worldwide, accounting for almost 20%of all global deaths.
As can be expected, most of the cases are in hospitals. In fact, a study found that almost 62%of the positive isolates from patients at ICUs with sepsis, were gram-negative organisms. There are several models, and some of the commonly used mice models of sepsis include LPS-induced endotoxemia, the cecal ligation and puncture model, and the monobacterial infection model systems.
In our laboratory, we have standardized a mouse model system, to induce peritoneal sepsis using Salmonella Typhimurium. This model is advantageous over others, as Salmonella Typhimurium is an intracellular pathogen that mimics the clinically relevant condition of gram-negative sepsis. The outcome of peritonitis sepsis in this model, is systemic with 100%mortality, within 96 hours post-infection.
Therefore, this model is instrumental in studying the inflammatory host responses. In this model, sepsis is induced by intraperitoneally injecting 0.5 million CFU of Salmonella Typhimurium, in an 8-10 week old C57BL/6 mouse. Systemic infection can be confirmed, by assessing organ bacterial burden, about 16 hours post-infection.
The experiments using Salmonella Typhimurium, requires BSL-2 facility, and adherence to BSL-2 guidelines. Care must be taken to use proper PPE, and to ensure safety, and follow standard BSL-2 biohazard disposal methods. All the experiments have been approved by the institutional animal ethics committee.
Culture preparation of Salmonella Typhimurium. Add a hundred microliter of Salmonella Typhimurium NCTC 12023 glycerol stock, into 3 mL of LB broth. Incubate the culture at 160 revolutions per minute, at 37 degrees celsius overnight.
Streak 50 microliter of the overnight grown culture in LB broth, onto a Salmonella Shigella agar plate, and incubate at 37 degree celsius for 12 hours. Pick a single colony from the streaked SS agar plate, using a microtip. Eject the microtip into 3 mL of LB broth, and culture at 160 RPM at 37 degrees celsius overnight.
Add 0.1 mL of the bacterial culture into 50 mL of LB broth, and incubate at 37 degrees celsius, in a shaker incubator at 160 RPM, for three to four hours, to reach the logarithmic phase of growth. Dilute the culture by a factor of two using LB broth. Measure the optical density of the culture at 600 nanometer wavelength of light in a spectrophotometer.
Once the OD reaches one, make two aliquots of 1 mL of culture, in 1.5 mL microfuge tubes. Centrifuge the tubes at 7, 750 G for 15 minutes. Discard this supernatant, and wash the pellet with 1 mL of 1XPBS twice.
Centrifuge the tubes at 7, 750 G for 15 minutes. Resuspend the pellet in 0.5 mL of 1XPBS, in two different 1.5 mL microfuge tubes. Combine the suspensions from both the tubes, into one 1.5 mL tube, now containing, approximately two multiplied by 10 raised to the power eight colony-forming units per mL.
Prepare a bacterial cell suspension of 10 power six CFU/mL, by diluting the stock solution. Important note, optimize the CFU corresponding to OD in your laboratory, to determine the CFU for OD one, before initiating the experiments. Mice and infections.
On the day of infection, hold the mouse with one hand, wipe the abdominal skin with 70%ethanol, and spread the hind legs for better accessibility of the abdominal wall. Inject 0.5 mL of 10 raised to the power six CFU/mL bacterial suspension intraperitoneally, with the help of a 1 mL syringe. Post-infection, plate the culture to check the actual CFU injected, which may vary from 0.2 to 0.8 million CFU per 0.5 mL.
CFU assessment of organs. The mouse was sacrificed using carbon dioxide asphyxiation. After sacrificing the infected mouse, wipe the abdomen with a piece of cotton, dipped in 70%ethanol.
Cut open the abdominal skin. Refer to the article by Ray and Dittle, for a video protocol, on how to collect peritoneal lavage fluid. Cut open the peritoneal cavity and collect the organ of interest.
In this video, we are demonstrating the enumeration of organ CFU from liver. The liver undergoes extensive histopathological damage, in this model of sepsis. Cut a small piece of liver and place it in a microfuge tube.
This can be stored in ice for two to three hours, before proceeding to the next step. Weigh the piece and transfer it into a microfuge tube. Preferably, cut the pieces weighing around 10 to 15 mg, for proper homogenization.
Add 0.5 mL of 1XPBS in the tube and homogenize the organs using a hand homogenizer. Make sure that the organs are completely homogenized. Make up the volume to 1 mL, by adding 0.5 mL of 1XPBS.
Centrifuge the tubes at 200 G for five minutes, at four degree celsius. Collect the supernatant into fresh microfuge tubes. Prepare dilutions of 10 to the power minus one, and 10 to the power minus two, in a 96 well plate.
Spread 50 microliter of the diluent, onto fresh SS agar plates, and incubate the plates at 37 degrees celsius for 12 hours. Count the number of colonies that appear in each condition, and normalize the data with the organ weight, using the following formula. CFU/mg is equal to number of colonies, multiplied by 20, multiplied by the dilution factor, the whole divided by organ weight in mg.
Note, the number 20 is used in the formula, to convert the colonies per plate to CFU/mL. This number is arrived at, by dividing 1 mL of the amount of a given volume of culture plated. In this case, 50 microliter.
For example, if you find a hundred colonies in an SS agar plate, where 50 microliter of 10 power minus one dilution of homogenized organ, weighing 10 mg is spread, then CFU/mg will be equal 100 multiplied by 20, multiplied by 10, the whole divided by 10, which is equal to 2000 CFU/mg. Flow cytometric analysis of various immune cell populations in peritoneal exudate. Collect the peritoneal cells as described previously, by Ray and Dittle.
Resuspend the cell pellet from peritoneal lavage fluid, in 1 mL of RPMI supplemented with 10%FBS. Enumerate the total cell numbers in the peritoneal lavage, using a hemocytometer. Adjust the cell number in such a way, that every tube receives two to five multiplied by 10 raised to the power five cells.
Spin the cells down at 200 G at four degree celsius for 10 minutes. Discard the supernatant. Wash the cells once with 1XPBS.
Centrifuge the cells at 200 G for 10 minutes. Block the Fc receptors using FcR blocker. One is to 400 dilution, prepared in blocking buffer, consisting of 5%FBS and 0.02%sodium azide in PBS.
Incubate on ice for 15 minutes. Centrifuge the cells at 200 G for 10 minutes. Discard the supernatant.
Dilute the fluorochrome conjugated antibodies of interest in blocking buffer. Here, we use one is to 500 dilution of anti-mouse Ly6G, to stain neutrophils. Note, other immune cell populations can also be shown, using anti-mouse B220 for B cells, anti-mouse CD3 for T cells, anti-mouse F4/80 for macrophages, et cetera.
Incubate about 0.2 million cells in 200 microliters of diluted solutions of antibodies, in separate tubes. As a negative control, set aside one tube in each fluorochrome type, for unstained control. In this tube, incubate the cells with 200 microliter of blocking buffer without antibody.
Incubate the samples on ice for 45 minutes, with intermittent tapping every 15 minutes. Centrifuge the cells at 200 G for 10 minutes, at four degree celsius. Discard the supernatant.
Fix the cells with 4%paraformaldehyde for 15 minutes at room temperature, if needed to store for several days. Resuspend the cells in 200 microliter of FACS staining buffer, 2%FBS in PBS. Acquire the data in the flow cytometer.
The images of SS agar plates shown here, indicate the organ CFU burden in liver and spleen of septic mice. The homogenized organ Lys6 were spread at dilution of 10 to the power minus one, and incubated at 37 degrees celsius. The black-pigmented S.Typhimurium colonies appeared after about 12 hours post-incubation.
A part of the plate is shown here as zoomed inset to highlight the colonies. These results suggest that the pathogen successfully disseminated systemically, and colonized the internal organs. This figure shows the sera isolated from healthy and septic mice.
The volume of collectable blood from septic mice is lesser than healthy controls, so the serum obtained is less. This happens because of heightened coagulation in sepsis. Also the sera from septic mice, show distinct red coloration, indicating the occurrence of extensive hemolysis.
The figure shows cytometric plots of peritoneal cells from healthy and septic mice, stained with anti-Ly6G antibody. These images are representative of one healthy and two infected mice. Septic mice saw an increased infiltration of neutrophils into the peritoneal cavity.
In this video, we have shown a method of inducing bacterial sepsis in mice, by intraperitoneal injection of Salmonella Typhimurium. This is a useful model to study the effects of therapeutic interventions in attenuating sepsis. In this model system, the cellular composition of the peritoneal cavity changes dramatically in order to combat the pathogen.
Therefore, it is a useful model, in studying the kinetic changes in immune cell compositions and functions during sepsis. The model is also useful in understanding the increase in intercellular reactive oxygen species, pro-inflammatory cytokine levels, hemolysis, and blood coagulation, all of which happen during sepsis.
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