A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

The cecal slurry model for polymicrobial sepsis is a powerful tool to dissect out unique neonatal pathophysiology. It can be variable, and humane endpoints aren't well described. Here we present our highly standardized procedure to prepare a cecal slurry and inject into experimental animals.

Furthermore, we use a data driven approach to identify moribund pups at an earliest possible time point. Let's begin by preparing a frozen cecal slurry stock. This is done by pooling cecal concent from at least five adult mice, suspending in sterile dextrose water, and storing at minus 80 for later use.

You can use males or females, but the sex, age, and background of mice for your experiments should be kept constant. First, let's prepare our tools for dissection. These include a set of scissors and forceps for the skin and a second set for the internal organs.

Turn on the hot bead sterilizer at least 30 minutes before use. Dip the tools in 70%ethanol and then submerge them in the hot bead sterilizer for at least one minute. Spray a mat of paper towels with 70%ethanol, then remove the tools from the sterilizer without touching the sterilized part of the tool to the non-sterile handles of other tools.

Place them on the paper towel to cool down. Dissect the mice in a biological safety cabinet. Pin the legs of the mouse to a styrofoam board using 23 gauge needles so the mouse is abdomen up.

Spray the abdomen with 70%ethanol. Using sterile forceps and scissors, cut through the skin, loosen the skin from the peritoneal lining with the scissors, and cut open a rectangular region from the groin to the sternum and left side to right side. Remove any fur around the peritoneum.

Switch to a new pair of sterile tools to cut through the peritoneum, also making a rectangular opening. Identify the cecum, which should be running left side to right side across the body. Disrupt connective tissue to identify the cecum branches from the intestines and cut the cecum away from the intestines.

Place it on a sterilized sheet of weigh paper. Using sterile tools, cut through both ends of the cecum. Hold the middle of the cecum with forceps and use a flat metal spatula to gently push the cecal contents out of the ends using a rolling motion.

Collect the content and place them in a pre-weighed 15 milliliter centrifuge tube. Pool cecal contents from a maximum of five mice into the same tube. Weight the tube again once all the contents have been added.

To calculate the volume of dextrose water to add to cecal contents in each tube, divide the weight of the cecal contents in the tube by your desired concentration in milligrams per milliliter. Keep an aliquot of dextrose water on ice for the re-suspension of cecal contents. Add the required amount of dextrose water to the 15 milliliter centrifuge tube containing cecal contents.

Vortex the tube vertically and horizontally for roughly 30 seconds. Observe for the presence of particulate over a few millimeters in diameter, and if present, continue vortexing until all the particulate has been visibly disappeared. Place a 50 milliliter centrifuge tube on ice to collect the filtrate and install a sterile 70 micrometer cell strainer over top.

Pipette four milliliters of re-suspended slurry from one 15 milliliter tube over the cell strainer and into the collection tube. Re-suspend the particulate by pipetting up and down two to three times. Gently extrude bubbles to increase filtering speed while stirring the contents with the pipette tip until there is no more liquid being filtered.

This should take roughly 30 seconds. Change cell strainers between each tube of cecal slurry, but pool all contents into the same 50 milliliter collection tube. If your stock exceeds 40 milliliters, then vortex it for 15 seconds, and place 20 milliliters into a new centrifuge tube.

Divide the slurry into cryogenic vials of 500 microliter aliquots each. Vortex the slurry between every three vials. Keep the master stock in vials on ice.

By storing aliquots at minus 80, the aerobic colony forming unit concentration in a cecal slurry preparation does not change over a six month period. Now that the cecal slurry is prepared, we can use it to challenge a litter of seven to eight day old mice with a weight-adjusted dose of cecal slurry. Lethality of the slurry is dose-dependent.

You will have to determine the desired lethal dose in your facility for every new stock with a new dose titration. One cecal slurry injection aliquot is prepared per litter. We normalize the inoculum to a 100 microliter volume, rounding to the nearest 10 microliters for each animal in the litter.

Preparing the challenge aliquot requires three calculations, the volume of slurry stock required, additional volume of dextrose water, and the rounded volume for each pup in the litter. Details on how to perform each calculation can be found in section three of the protocol. Performing these calculations by hand for every experiment can be time consuming and will contribute to experimental error, thus a spreadsheet can be downloaded along with the protocol where you input your stock concentration, desired dose, and weight of your litter, and the amount of slurry stock, volume of dextrose water, and actual volume per pup is automatically returned.

You can print this sheet and take it with you when you're ready to challenge your animals. Prepare the injection aliquot in a biological safety cabinet and keep it on ice. Before loading the syringe, mix the microcentrifuge tube by flicking 20 times.

Pups are challenged with a 28 gauge insulin syringe. Before drawing up slurry, mix the aliquot by inspiring and ejecting 300 to 500 microliters of the challenge aliquot in and out of the microcentrifuge tube. Then, draw up roughly 150 microliters of the challenge aliquot.

This should be larger than the injection volume for any seven to eight day old pup. Flick at the syringe to dislodge bubbles from the plunger and draw back slightly on the syringe to expel any air bubbles. Do not touch the needle or the plunger shaft during this process.

Dispense the excess cecal slurry back into the microcentrifuge tube until the calculated amount of cecal slurry for the mouse is loaded into the syringe. Cecal slurry is administered with an intraperitoneal injection. Perform this according to the protocol at your animal care institution.

To minimize leaks, tilt the pup to face downwards and insert the needle between the leg and the genitalia, keeping the needle shallow and subcutaneous. After inserting the needle about one centimeter, press downwards and forwards until you feel the needle puncture the peritoneum, then slowly depress the plunger. Carefully withdraw the needle following the same route as you went in, relaxing your middle finger as you do so.

Note any leaks in your lab notebook and consider omitting animals with substantial leaks from your final result. In this section we will outline the different levels of health that we observe in mice challenged with polymicrobial sepsis and their corresponding humane endpoint. For any procedures involving neonatal mice, we transfer the bedding material to a new cage.

This transfers the dame's smell onto your gloves and reduces the stress on the dame from repeated entry into her cage. We make a second nest in the new cage to hold the monitored pups until the end of the procedure or monitoring when the entire litter is ready to be returned to the dame's cage. Place a mouse on its back and monitor for the ability to right within four seconds.

If it can right, then wait eight more seconds to determine the level of mobility. An unchallenged or recovering mouse is most often able to right and explore its environment by taking multiple steps in a row. Regroup mice who are able to righten themselves and take a few steps to explore its environment as rights-lethargic.

These mice may fall over while taking a step and look shaky on their feet. A mouse which is unable to right and has hip movement that exceeds 90 degrees from horizontal we group as fail to right-mobile. This mouse will eventually right but cannot within four seconds.

Because of its hip movements, it is grouped as fail to right-mobile. Pups that were unable to right and have hip movement below 90 degrees from horizontal are grouped as fail to right-lethargic. This is another example of a mouse whose hips do not exceed 90 degrees from horizontal and is grouped as fail to right-lethargic.

A fail to right-nonmobile pup will have legs that may shake or vibrate but have no hip movement. Limbs may extend and retract but will not have lateral movement. The pup is visibly ill and has reached the humane endpoint.

This is another example of a fail to right-nonmobile pup. Its hips do not shake left to right, but its paws do vibrate. When handling the dame's cage, pups can be dragged out of the nest if the nursing dame runs.

We replace these and then check for other scattered pups which are away from their nestmates. If those scattered pups are fail to right-lethargic, they are at a humane endpoint. Here's a Kaplan-Meier survival curve of our sepsis model.

The monitoring time points can be found in the attached SOP. At any point along the survival curve if a mouse is found to be fail to right-nonmobile, it is at its humane endpoint and euthanized. Between 12 and 21 hours post-challenge, mice will appear sick and about half of the mice will be able to right on one side, while the other half fail to right on either of their sides.

This is the timeframe where the mice will begin to digress in health score and reach the humane endpoint of fail to right-nonmobile. These are representative results of health scores observed in this timeframe. The survivor and non-survivor groups cannot be differentiated at this point based on their health scores.

In this stage of disease, 21 to 48 hours post-challenge, we observe the highest proportion of humane endpoints. We have also observed that the vast majority of any mice that are unable to right on both sides will not end up recovering from disease, which therefore becomes a new criteria for humane endpoint in addition to the fail to right-nonmobile criteria. The observed health scores during this stage of disease resulted in a separation of survivors from non-survivors.

The vast majority of mice that were unable to right on both sides did not recover from disease. This data supports that a new criteria for humane endpoint beginning at 21 hours post-challenge should be to euthanize any mice that fail to right on both sides. Over 48 hours post-challenge many mice will begin to display more activity and a better righting reflex.

Even though the proportion of humane endpoints are lower in this period, the mice can still digress and become sicker, and they still require monitoring for humane endpoint. During this timeframe, some of those mice that were eventually grouped as non-survivors had displayed increased activity at one point but later did worse and reached a humane endpoint. This necessitates continued monitoring of sick animals with increased frequency if they begin to display worse health outcomes.

Independent experimenters that have not received in-person training and that only observe this instructional video were tested in their ability to assign the same score as the investigators using recorded test videos. This instructional video results in accurate behavioral classification. The ability to assign the correct score to fail to right-nonmobile neonatal mice, which is a measure of humane endpoint, was determined out of 60 neonatal mice, and an average of 97%of behaviors were accurately distinguished, with only 3%being misidentified.

Next, the ability to differentiate neonatal mice that were fail to right or that were able to right within four seconds was tested. This is another measure of humane endpoint and was accurately assigned in 97%of neonatal mice, while 3%of pups were incorrectly assigned as righting. Here are representative results for the percent weight change of mice compared to their time of challenge.

The average weight change between survivors and non-survivors began to separate at 24 hours post-challenge, but there is a lot of overlap between the groups. There were some survivors that lost over 20%weight but still recovered from disease. Weight change was not as efficient in predicting outcome as compared to the differences in health scores which is why it is not used as a criteria in determining whether mice are at a humane endpoint.

To conclude, you should now be able to prepare a stock of frozen cecal slurry, calculate the weight-adjusted dose. And inject and monitor newborn mice for humane endpoints. We hope this will be a valuable resource for your mouse research.