Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Prepare the mouse for surgery. Shave the lower quadrants of the abdomen using a razor and disinfect the area with iodine. Place the mouse onto a clean work surface on its back with the head pointing away from the operator.

Make a longitudinal skin incision about a half a centimeter long in the left lower abdomen. Be careful not to penetrate the peritoneal cavity. Use small scissors to extend the incision about one to two centimeters.

Make an intramuscular incision to gain access into the peritoneal cavity. In most cases, the cecum is on the left side of the abdomen. Isolate the cecum.

Using blunt, anatomical forceps, gently and carefully expose the cecum through the incision. Step four is the ligation. The position of the ligation on the cecum determines the severity level of sepsis.

Ligating approximately 75%of the cecum induces severe sepsis. Ligating 50%of the cecum induces mid-level sepsis. Whereas ligating 25%or less of the cecum will result in minor sepsis.

Ligate the cecum at the designated position. Make sure not to ligate the ileocecal valve. Step five is to puncture the cecum.

Using a needle, perforate the cecum with a single through and through puncture mid-way between the ligation and the tip of the cecum. Make sure you do not puncture any blood vessels. To ensure that there was a complete puncture, extrude a small droplet of feces from both needle holes.

Carefully replace the cecum into the abdominal cavity. Close the abdominal musculature by applying simple running sutures and close the skin. Clean the skin with iodine.

Inject the mouse with a pre-warmed saline solution to resuscitate. Place the mouse back into the cage with unlimited access to food and water. After about three days from the cecal ligation and puncture or the CLP procedure, we subject the mouse to a second hit with an intranasal infection to stimulate pneumonia that often occurs in sepsis.

Approach the neck of the mouse from behind, smoothly and gently. The thumb and index finger of the left hand form a U-shaped position. Hold the neck immediately behind the ears so the mouse head is completely restrained.

Introduce the intranasal infection. Using a 100 microliter pipette, instill the staphylococcus aureus strain intranasally. Holding the mouse in an upright position, slowly drop two to three microliters of the bacterial solution into both nostrils of the mouse.

Raise the mouse up and down with its head up and tail down to help the bacterial solution enter the trachea. Raise the mouse up quickly while lowering it gently and slowly. Repeat the instillation 15 times or until all the bacterial solution is instilled.

The last step is to finish the procedure. Lay the mouse on the angled bedding at about a 35 degree angle to recover. Make sure the mouse is on its back with its head laying up.

Watch the mouse to make sure it is completely recovered from the anesthesia and the procedure. After flattening the bedding, place the mouse back in the cage with unlimited access to food and water. Mice began to die at around 12 hours after induction of peritonitis.

The mortality was much higher for mice with CLP before a pneumonia. Three days post-surgery, CLP mice had more mortality with an intranasal administration of staphylococcus aureus. The levels of serum pro-inflammatory cytokines significantly increased at 24 hours after bacterial instillation in sepsis mice compared to the mice that had CLP alone or the Sham operated mice with a second hit.

However, double-hit mice exhibited significant decrease in BAL fluid cytokine levels compared to both SA and Sham surgery plus SA mice. Hence CLP impairs host defense and increases susceptibility to infection. This dual-hit model is a valuable tool to understand the molecular mechanisms for host-pathogen interaction, which may open the avenue to develop new, effective drugs and other therapeutics for superbug infections.