The overall goal of this centrally categorized mouse method for cecal ligation and puncture, or CLP, is to create a reproducible model of prolonged critical illness with intensive care measures to mimic the human clinical setting. This method can help answer key questions in the fields of intensive care medicine. About a pathogenesis of endocrine and metabolic alterations during acute and prolonged critical illnesses.
The main advantage of this technique is that it combines the well standardized CLP technique with the use of a central venous line for fluid, drug and nutrient administration. Demonstrating the procedure will be Thomas Dufour, a technician from our lab. To prepare the venous line tip, quickly immerse the middle section of a piece of 60 centimeter micro-renathane tubing in 220 degree celsius or hotter sesame oil.
Gently pull on the ends of the tubing to stretch the middle of the tube, to produce a narrow central diameter and use a scalpel blade to cut the tubing into two equal 30 centimeter pieces. Now, connect the tubing to a polyethylene PE 50 connector, a swivel and a Luer stub needle, to construct the catheter as demonstrated and apply strong fast acting adhesive glue to all of the connections. When the glue is cured, flush the catheter with air, to check for leakage, next, cut an 80 centimeter piece from a 0.8 millimeter diameter metal wire, and fold the wire back on itself to make a loop.
Lead three pearls over the doubled wire of the loop, and guide the tip of the sterilized catheter through the three pearls, to attach the catheter to the wire. Anaesthetize a 27 to 32 gram, 24 week old male c57 black six mouse, and confirm sedation by a lack of response to toe pinch. Use forceps to gently pull the tongue out of the mouth, to avoid swallowing the tongue.
Next, shave the surgical area, then place the mouse in the prone position on a pre-warmed heating pad. Disinfect the exposed skin with 70%ethanol and apply a small amount of ophthalmic lubricant. Infiltrate the surgical sites with analgesia and make a small incision at the base of the dorsal side of the head.
Expose the posterior cervical muscles, and thread a piece of 3.0 nylon under the muscles to bind the loop of the attachment wire to the muscle. Next, with the mouse on its right side, make a small vertical incision in the skin of the ventral neck. Tunnel an 18 gauge needle subcutaneously through this ventral incision toward the incision on the back and thread the catheter through the needle to exteriorize the needle at the ventral side.
With the mouse on its back, pass a three zero nylon thread behind the top incisor teeth and tape the nylon to the heating pad. Then, secure the tail and forelimbs in a stretched position. Now, focus the dissecting microscope and use forceps to gently tease away the fat tissue and glands in the ventral incision until the jugular vein can be visualized.
Place the tip of the forceps between the vein and connective tissue, and repeatedly open the forceps in parallel with the vein to bluntly dissect the jugular vein free from the connective and subcutaneous tissue around the vessel. When the vessel is isolated, place the blunt forceps under the jugular vein and feed two pieces of three zero silk thread under the vein. Positioning one piece, approximal to the bifurcation of the jugular vein for the cranial ligature and one piece close to the sternocleidomastoid muscle for the caudal ligature.
Tighten the cranial and caudal ligatures, to stretch the vein and to prevent excessive bleeding while placing the catheter. Use a third thread to create a loose ligature, then, using micro-scissors, make an incision along the vein between the cranial and caudal ligatures large enough to pass the catheter. Using forceps, insert the catheter 11 millimeters into the vein and loosely secure the catheter with the middle ligature.
Gently flush the catheter with sterile saline to confirm its correct placement. Then, firmly tighten the caudal and middle ligature. Tie the ends of the caudal and middle ligatures together to secure the catheter.
Confirming that the vein is not occluded by the knots, by flushing the catheter after every tie. Finally, ligate also the cranial ligature. Then, close the incision with five zero silk sutures.
For Cecal Ligation and Puncture, make a one centimeter midline incision through skin of the lower half of the abdomen, taking care not to penetrate the peritoneal cavity. After identifying the linea alba of the abdominal musculature. Make an inter-musculature incision to gain entry into the peritoneal cavity.
Using blunt anatomical forceps, isolate and exteriorize the cecum. Ligate the cecum at 50%of its length with three zero silk sutures, taking care not to constrict the ileocecal valve, so that the intestinal continuity is maintained. Then, use an 18 gauge needle to perforate the cecum with a single through and through puncture midway between the ligation and the tip of the cecum, extruding a small amount of feces from the hole, to ensure patency after removing the needle.
Reposition the bowel in the abdominal cavity, and close the peritoneum and skin with five zero silk sutures. With the mouse in the supine position, tape the catheter to the attachment wire. Then, place the mouse in an individual cage with nesting material and a wooden block.
Using a stand with an adjustable clamp, place the swivel device in the clamp and firmly tape the free part of the metal attachment wire to the rotating point of the swivel. Then, place the cage in a 27 degree celsius temperature controlled animal cabinet with 12 hours light and dark cycles. Attach the syringe containing the appropriate mixture of balanced colloids and crystalloids into the venous line and use an accurate syringe driven infusion pump, to start the fluid resuscitation at 0.3 millimeters per hour.
After six hours, inject analgesia and antibiotics. Analysis of the survival curves of one five day, and one seven day cecal ligation and puncture experiment, reveals that the survival rates were not significantly different between the two studies up through day five, underscoring the reproducibility of the experimental set up. The ligation of 50%of the cecum in combination with antibiotics, fluid resuscitation and total parenteral nutrition via a venous catheter in the vena jugularis, results in a 13%mortality after one day of critical illness.
24%mortality after three days of critical illness, and 27%to 31%mortality after five days of critical illness and 36%mortality after seven days of critical illness. The healthy para fed mice that were caloric restricted to the nutrient intake of the critically ill mice, however, did not demonstrate any mortality in either experiment. After 10 to 15 animals to master the technique, the catheter can be placed and CLP can be performed over a period of 45 minutes, if the procedures are performed properly.
When performing this procedure, remember to place the catheter into the vein carefully. Placing the catheter too far into the heart, will induce heart arrhythmias. Placing it not far enough, it can be pulled out easily.
Always confirm that the catheter is placed within the lumen of the vessel. If the catheter is placed within the layers of the vessel wall, fluids will leak out into the surrounding tissues. This model may require an extensive surgical procedure and daily intensive care of the animal.
But it results in a stable and reproducible model which allows the study of many aspects of acute and prolonged critical illnesses.
