Hello, my name is Miguel Toscano from Dr.Dena's lab in the department of Microbiology and Hemology at Temple University. Today we are going to show you how to set up the iCal allegation and puncture model in the C 57 Black Seas Mouse strain. We use this model in our laboratory to mimic human sepsis.
Basically, this model consists in a puncture of the seum that allows the release of fecal material into the peritoneal cavity to produce a polymicrobial sepsis, thus generating an exacerbated immune response. In this model, many organs and different cell types are irreversibly damaged. That in the end can cause the death of the animal.
So let's get Started. Anesthetizing the mouse. The first step is anesthetizing the animal by injecting intraperitoneal.
A mixture, one to one of Ketomine and xylazine. To avoid being bitten by the mouse, to avoid hurting him, we must grab the mouse from the tail with one hand. With the other hand, we presses back against the floor.
Then we can grab the mouse from the back of the head and then turn the animal on his back to expose the abdomen. With an insulin syringe of 29 gauge, we inject 20 to 30 microliters of the anesthesia in the peritoneal cavity. Preparing the mouse for procedure, grab the back of the mouse and shave its abdomen By pressing the tail and or the leg of the mouse.
We check that the mouse is not sensitive to pain, Then the abdomen is Disinfected. First by scrubbing with Betadine solution, followed by wiping with an isopropyl alcohol wipe For at least one minute. Practicing the laparotomy To practice a laparotomy, we lift the skin in the middle of the abdomen with the forceps and making a small incision of about one centimeter In length.
Then Lift the perineum and practice another small incision to expose the peritoneal cavity. Using curved Forceps, hold the peritoneal membrane. Identify and expose the cecum by passing it through the peritoneal membrane.
Place it over the abdomen. Now is the moment of the ligation. This is an important factor.
In order to determine the severity of the model, we can modulate the severity of the procedure. By the extension of the ligation, mice will develop a milder sepsis. If ligation is made closer to the end of the cecum, a mid-grade sepsis if done in the middle and a severe sepsis if done closer to the ileocecal valve, the severity of the procedure will Be reflected by the percentage of mortality.
Never ligate the ileocecal valve. Now make the ligation using silk suture with a double knot to make it tight, making the puncture Hold the cecum and carefully make a puncture close to the ligation site and make another puncture closer to the end of the cecum. The membrane of the secum is soft and it'll be perforated very easily.
Using needles with different thicknesses and making one or two perforations, we also can modulate the severity of the model. The the single or double puncture method shown here is an alternate technique to the classical through and through sequel puncture. The advantage of using the single or double puncture method is that one can control better the amount of feces extruded when they seek em is squeezed.
Hold the secum with both fingers and gently extrude the cecum to extract a very small amount of feces. Closing the laparotomy, Then lift the peritoneum and carefully place back the cecum. In the peritoneal cavity.
Be extremely careful to avoid accidental pricks of the cecum With the forceps, the use of bigger forceps Is recommended. Now is the time To close the peritoneal cavity. Using silk suture, it is very important that you make sure it is very well closed.
We start from one side of the wound where we make a knot. Keep making the suture until the other side by passing the silk under the loop of the Previous puncture. To close the skin, we can use Mitchell wound clips or reflex clips using the right tool to close the clips.
Place as many clips as needed to make sure the wound is Closed. Resuscitate Mice by injecting subcutaneously, one milliliter of prewarm saline solution. Using a 25 gauge needle, this fluid resuscitation measure will induce the hyperdynamic phase of sepsis, finishing the procedure.
Place the mouse under an infrared heating light, or alternatively over a heating pad Until the mouse recovers from the anesthesia. Provide food and water on the floor of the cage and supervise the animals every eight to 12 hours for signs of disease. Inject buprenorphine Subcutaneously for postoperative analgesia.
A different analgesic to use is T.Tramadol. Signs of sepsis, symptoms of sepsis are fever, lethargy, pelo, erection, diarrhea, huddling, and malaise. Very sick animals show a severe decrease in body temperature and they barely move to avoid suffering.
They should be euthanized at this stage H.So this is how we perform the sickle ligation and punctual model. Hopefully, you have enjoyed the demonstration of this procedure and found it very useful for your research before starting your animal study. We strongly recommend you using old or unused minds for training in order to establish the appropriate degree of severity for your experiments by modifying the length of the ligation, the thickness of the needle, and the number of punctures.
With that said, goodbye.
