The overall goal of this procedure is to generate abdominal sepsis by inserting a stent into the ascending colon of a mouse. This is accomplished by first preparing the ascending colon. Next, an adapted cannula is inserted into the colon and then fixed in place.
Finally, the stent is removed and the colon is placed back into the peritoneal cavity. Ultimately, results can be obtained that show sepsis by measuring cytokines and bacterial counts in plasma. The main advantage of this technique over existing methods like sickle lation and puncture, is that epsis and mortality can be titrated by the size of the inserted stand.
So generally, individuals who are new to this method, they could struggle because microsurgical techniques, they need a bit practice. Until perfect performance can be provided, Begin by anesthetizing the mouse with an intraperitoneal injection of narcotic fluid and place the animal in a supine position. The feet of the mouse need to be fixed with tape on the plate to ensure a stable position of the animal during the operation.
For this procedure, adapt an IV cannula, which is commonly used for IV injections in humans and carve its plastic tubes. Circularly two millimeters from its tip after thorough disinfection of the abdominal skin of the mouse, incise along its midline about 15 millimeters. Open the peritoneal cavern by incising the abdominal muscles and the peritoneum along the linear alba.
Identify the sequel pole and use cotton swabs to carefully pull the secum terminal ileum and ascending colon out of the abdomen. 15 millimeters distal from the ileocecal valve pierced the wall of the ascending colon with a seven oh suture. Fix the suture on the colonic wall with two surgical knots.
Puncture the ascending colon with a prepared cannula. One to two millimeters proximal from the seven oh suture. Carefully insert the cannula into the colon until the furrow and the plastic tube is on a level with the cirr.
Put the free ends of the seven oh suture around the cannula and place a twofold knot exactly into the prepared furrow of the plastic tube. Take the needle of the seven oh suture and stitch it through the anti mesenteric wall of the colon consecutively. Perform two surgical knots to additionally fix the plastic tube and the colonic wall.
Cut the suture ends. Retrieve the iron part of the cannula a little, and cut the plastic tube off close to the fixing 7.0 suture. Now carefully milk stool from the cecum towards the colon stent by using cotton swabs until a small tip of stool appears on top of the stent.
Put the gut back into the peritoneal cavern and perform fluid resuscitation by intraperitoneal administration of 0.5 milliliters of saline solution. Close the peritoneum with a continuous suture. Close the skin with singular sutures for analgesia.
Subcutaneous administration of a powerful analgesic substance like buprenorphine should be performed if necessary, put the animal back in its cage, which should contain food and water. Five hours after casp, prepare and anesthetize the mouse again to perform the operational intervention on the animal. Open the sutures of the abdominal wall.
Pull out the ascending colon with the stent inserted carefully cut the sutures fixing the stent and remove the stent. Close the defect in the colon with single inverting sutures. Put the gut back into the abdominal cavity and flush the ladder twice with five milliliters of saline solution.
Close the peritoneum with a continuous suture. Close the skin with singular sutures for analgesia. Regularly perform subcutaneous administration of a powerful analgesic substance.
Place the animal back in its cage with ample food and water within the first two days after the operation. Monitor the animals every six hours within a few hours following the CASP operation, animals show clinical signs of a beginning sepsis survival rates following CASP surgery depend on the diameter of the inserted stent. 14 gauge CASP results in a mortality rate of 100%16 gauge CASP results in 70%mortality and 18 gauge CASP leads to 50%mortality.
Sham CASP is associated with a survival of 100%The outcome of CASP by depends on the period of time. After casp, when the stent is removed, an intervention three hours after CSP results in a survival rate of 45%If the stent is removed five hours after csp, only 10%of the animals survive the procedure. A stent removal after nine hours results in a mortality of 100%sham CASP by leads to 100%survival.
Macroscopic inspection of the abdominal sitis 24 hours after CSP shows typical signs of peritonitis, edema, vasodilatation, arithmatic, gut wall sharp demarcation of pires patches, gut paralysis, free fluid and septic secretion. The systemic infection can be shown by bacteriological analysis 12 hours after csp. Massive bacterial amounts can be detected in peritoneal lavage, blood, liver, lung, spleen, and kidneys.
CSP induced sepsis manifests in local and also systemic release of pro and anti-inflammatory cytokines and chemokines. 12 hours following csp, significant amounts of T-N-F-I-L one beta IL six IL 10 MCP one and others can be measured by Eliza in blood, but also in supernatants of organ suspensions such as liver, lung, spleen, and kidney. Once mastered, this technique can be done in 10 minutes if it is performed properly.
After watching this video, you should have a good understanding of how to generate abdominal sepsis with the models of SSP and SSP with intervention. That was it. Thank you for watching and good luck with your experiments.
