Repeated blood sampling from the small laboratory animal is an important aspect of pharmaceutical lead optimization. This protocol details the microsurgical skills to establish a permanent jugular vein cannulation rat model. This technique can efficiently establish a jugular vein cannulation rat model and repeatedly collect blood samples through the cannula in conscious rats while avoiding stress and pain.
It's important to locate the jugular vein and its cannulation as the visual demonstration of anatomical complexity is not available for this model. To begin, prepare the aseptic workstation with 75%medical alcohol, then anesthetize the shaved rat with isoflurane mixed with oxygen. Subcutaneously inject meloxicam solution at a dose of two milligrams per kilogram, then carefully lift the skin near the clavicle on the right side of the midline of the neck with forceps, and using a pair of surgical scissors, make a 1.5 to 2 centimeter long incision towards the chest.
For exposing the underneath jugular vein, use iris scissors to blunt dissect the thin tissue cover. The proximal cephalic end of the external jugular vein consists of two branches which can be visually identified. Lift the jugular vein along with its connective membraneous tissue to visualize the lymph gland attached to the jugular vein.
Carefully separate the vein along the vascular direction from the surrounding muscle, fat, and other tissues. Nudge the forceps under the jugular vein without damaging the collateral blood vessels and pass two pieces of 6-0 suture under the vein to mark the two ends of the blood vessel individually. Pull one piece of the suture as far as possible toward the rat head and ligate the vein cranially with two to three knots using forceps.
Place the second ligature on the caudal end of the vein with one loose knot. Attach an 11 centimeter polyurethane, or PU, catheter to the prepared blunt tip syringe filled with the heparinized saline, and slowly push the heparinized saline into the catheter to avoid air bubbles. Nudge the non-tip flat side of the forceps under the jugular vein to exit on the other side.
Make a small V-shaped cut on the vein near the cranial tie with a pair of castroviejo microscissors, and gently opened the incision with the tip of the elbow vessel dilator forceps. Cut out the oblique opening of the front end of the jugular vein catheter. Plant the oblique end of the tube with forceps and slide it into the jugular vein.
While advancing the catheter, slowly withdraw the elbow microsurgical forceps, and clamp the outer surface of the vessel with forceps. Stop inserting the catheter upon hitting the first blue mark of the PU tube, which is approximately three centimeters in length. Secure the inserted catheter to the vein with both caudal and rostral ligatures using forceps.
Thread a 6-0 suture through the exposed tissue on the right side of the incision using a suture needle, and tie the ligature with a hemostat. Then bend the catheter at the second blue mark to bind with the same ligature and avoid occluding the PU tubing. After snipping all the extra suture thread, close the catheter by replacing the blunt tipped syringe with a 22 gauge stainless steel plug.
Place the rat in the dorsal position and gently clean the area between the scapula with the cotton ball soaked in 75%medical alcohol. Using the surgical scissors, make a very small incision at the center of the dorsal neck. Through the dorsal incision, guide and gently push the trochar underneath the skin toward the ventral incision on the right side of the neck.
Put the venous catheter into the trochar and then pull out and guide the venous catheter toward the dorsal incision. After securing the exteriorized catheter into the muscle layer, close the skin layer of the ventral and dorsal incisions with the 6-0 nylon suture and suture needle and swab all surgical incisions with iodophor. Next, remove the catheter plug by clasping the catheter with fingertips.
Place a new blunt tip syringe and slowly draw back the syringe to test the blood flow. Hold the catheter again with fingertips. Inject 0.2 milliliters of heparinized saline and 0.1 milliliters of lock solution into the catheter using the blunt tip syringe, and then replace the syringe with a stainless steel plug.
Unclamp the catheter and push the plug in slightly to ensure the tightness of the catheter. To collect blood samples for hematological tests, place the rat in a restrainer, open the plug, and insert the syringe into the venous PU catheter to ensure the catheter is not obstructed. Discard the initially withdrawn blood containing a mixture of blood, heparinized saline, and catheter lock solution.
Using a new syringe, collect 150 microliters of fresh blood sample and transfer the blood sample to a 0.5 milliliter tube previously sprayed with K2EDTA. Inject 150 microliters of pre-warmed normal saline, and infuse 0.2 milliliters of sterile heparinized normal saline through the catheter. Inject 100 microliters of the lock solution into the catheter to ensure the sealing and sterility of the catheter before the next sample collection.
In the current study, the physiological and hematological conditions over six days postoperatively were investigated. The majority of the rats recovered within four to six days post surgery as evidenced by body weight gain of more than 10 grams, regular food intake, selected blood components relating to infection, dehydration, and inflammation, including white blood cell, red blood cell, hemoglobin, and platelet count. Thus, the rats required a minimum of four to six days for recovery after the procedure.
The large intake of water indicated dehydration on the first day post-operation. In addition, the pharmacokinetics of the natural polyphenol ellagic acid was studied in the established jugular vein cannulation rat model and the poor bioavailability of the ellagic acid was indicated by its low plasma concentration over 24 hours. Correct isolation of the jugular vein is the most important first step because the jugular vein embedded within other soft tissues may not be immediately visible to the researcher.
Sequential blood sampling within the same animal can be performed in the established JVC rat model. It's a useful and robust tool to evaluate drug formulations performance in vivo.
