This protocol is robust and reproducible model of endotoxemia in which physiological and molecular parameters can be assessed repeatedly. This approach may help to answer questions in sepsis research. While our model does not replace animal experiments, it refines sepsis models through continuous sedation and reduces the number of animals through repeated sampling.
Beginners might struggle with the insertion of arterial and venous catheters, but only limited training is required to master these techniques. Visualization of the entire experimental setup facilitates researchers to set up, or adapt, their models to mimic more closely a situation on ICU. Begin by transferring the anesthetized animal to a heating mat.
Keeping the body temperature between 36.5 and 37 degrees Celsius. Make sure that the animal is spontaneously breathing by providing oxygen with a nose cone. Confirm the level of anesthesia by the absence of the toe pinch reflex prior to the installation of tracheostomy and arterial and venous catheters.
Use an ointment to protect the eyes. Prepare sterile surgical instruments and catheters along with pressure and oxygen saturation monitoring for sufficient oxygenation. Apply a tourniquet at the rat's proximal tail to facilitate venous access and disinfect the tail three times with alcohol.
Introduce a 26-gauge intravenous catheter into one of the two lateral tail veins avoiding air injection. Untie the tourniquet after placing the intravenous catheter and fix the intravenous catheter in place with adhesive tape. Connect syringe pumps with three-way stopcocks to the intravenous access for continuous fluid and drug application.
Begin the tracheostomy by shaving the animal's anterior neck area, and then disinfect the shaved skin three times with povidone iodine solution. Perform a two centimeter longitudinal incision using a scalpel blade number 10. Retract the skin with 2-0 silk sutures.
Then bluntly prepared the larynx and the trachea to be opened with surgical scissors. Insert a sterile tracheal cannula into the trachea, being careful not to penetrate too deeply in order to avoid unilateral ventilation. Fix the cannula in place by using a 2-O silk suture and then connect the cannula to a ventilator for pressure or volume-controlled ventilation.
Define fluid replacement protocols, vasoconstrictor application protocols, and abortion criteria before setting up the experiment. Disinfect the rat tail three times with povidone iodine solution and then cut the skin circle one centimeter longitudinally at the ventral side using a scalpel, taking care not to cut too deeply to avoid an injury of the tail artery. Use a surgical microscope to expose the artery carefully.
Cut the fascia surrounding the artery with surgical micro scissors. Then ligate the distal part of the artery by performing proximal 6-0 silk suture, but do not tighten the silk. Introduce a 26-gauge catheter into the artery between the distal and proximal silk suture, and then tighten the proximal silk suture to fix the catheter in place.
Connect the catheter to a pressure transducer to provide continuous arterial pressure measurement. Additionally, place a three-way stopcock between the catheter connected to the pressure transducer and the 26-gauge catheter for arterial blood sampling. After the animal has reached a steady state, induced sepsis by injecting LPS and collect blood samples.
Replace fluid loss from the blood samples by Ringer's solution at a ratio of one to four, avoiding air injection at all times in order to prevent air embolism. The mean arterial pressure remains stable in animals with and without LPS stimulation. LPS-treated animals develop characteristics of sepsis, such as negative base excess.
LPS-treated animals also developed a strong inflammatory reaction, which was measured by plasma cytokines, such as chemoattractant protein-1, monocyte chemoattractant protein-1, and interleukin-6. When attempting this protocol, remember to predefine fluid and vasopressor regimens as well as to define termination criteria in order to obtain solid and reproducible research data. This protocol can be adapted to other sepsis models.
So a research question is answered with the most suitable approach and in accordance with the principles of reduce, refine, replace of animal welfare.
