The overall goal of this procedure is to catheterize the jugular veins and carotid arteries of rats to infuse glucose and lipids as a model for chronic nutrient excess. This is accomplished by first catheterizing, the jugular vein and carotid artery under general anesthesia. After the procedure, the rat is allowed to recover for six days.
Then the catheters are connected to infusion pumps through a tether and swivel mounted on the cage top. Finally, the rat is infused with glucose and lipid solutions. Ultimately, results can be obtained that show the effects of chronic nutrient excess on glucose homeostasis.
The main advantage of this technique over other models, such as genetic models, for instance, is that one can examine the early changes in beta cell function in response to nutrient excess. The other advantage is that one can specifically modulate the levels of glucose and intralipid or lipids in the circulation. Now, we use this technique to examine changes in beta cell function, but of course, it can be used to look at other physiological parameters such as insulin resistance.
For example, demonstrating the procedure will be Grace Ferguson, an animal health technician in my laboratory, Prior to beginning, sterilize all surgical instruments by autoclave at least 16 to 24 hours before the procedure. Place the cannulation tubing into 2.6%glute aldehyde for sterilization. Using four rats per infusion condition, weigh each rat and then calculate dosages for the analgesia and anticholinergic drugs.
After anesthetizing the rat with two to 3%iso, fluorine and oxygen, check the level of sedation by toe. Pinch and place the rat on its stomach. Shave the area behind the ears to the base of the shoulders, then turn the animal on its back, and shave the region under the neck to the front paws.
Next, disinfect the surgical site three times with chlorhexidine alcohol and iodine, and then administer carprofen and glycopyrrolate. Transfer the rat to the surgical area, and then with aseptic technique. Using a PE 50 cannula attached to a one milliliter syringe filled with five units of heparinized saline, cannulate the right jugular vein and left carotid artery To avoid clotting during the recovery period, flush the cannulas with 50 units of heparinized saline through a blunted 23 gauge needle, and then use another 23 gauge pin to close each cannula after the surgery, trim about 2.5 millimeters off the bottom of the incisors, and place the rat in an infusion jacket to prevent the cannulas from being eaten.
To help evacuate the ISO fluorine, administer oxygen at one liter per minute. Place the rat in a heated cage until it is fully awake. On days one and two, post-surgery.
Weigh the rats, administer glycopyrrolate, subcutaneously twice on day one and once on day two, and provide additional treatments when necessary according to the text protocol on day seven post-surgery. After weighing each rat to calculate the flow rates for infusion, connect the cannula of the jugular vein by a tubing extension inside of the spring to the blue swivel. Flush the cannulas with five units of heparinized saline and connect them to the tether.
Then connect the rat to the infusion system using the tether and swivel. Now flush the cannulas once more with five units of heparinized saline to prevent clotting. Allow the rats to acclimate to the tether and swivel for at least 24 hours before starting the infusion to carry out the infusion.
Begin by drawing 0.15 milliliters of blood from the carotid of each rat and measure glycemia. Flush the jugular cannulas and use 50 units of heparinized saline to prevent clotting of both cannulas. Transfer the blood sample to a 0.5 milliliter collection tube containing 2%EDTA, then centrifuge at 10, 000 RBM for two minutes, and freeze the plasma at minus 20 degrees Celsius.
Fill two 60 milliliter syringes for each of the infusion conditions listed here. Use y connectors and sterilized coex T 22 tubing to join each pair of solutions together. Then place the syringes on a Harbor 33 to syringe pump placing syringe one on the front position of the pump and syringe two on the back position.
Next, change the cage bottom and remove all food from the cage grill top before replacing it with 150 grams of standard chow. Then connect the syringes to the swivel on the cage grill and flush the syringes properly. To remove trapped air from the lines using the most recently determined body weight, calculate the infusion flow rates using an Excel file that converts the glucose infusion rate or GIR into milliliters per hour based on predetermined glucose levels desired to maintain throughout the experiment.
Set the pump to administer flow rates for 60 syringes according to the manufacturer's settings, and enter the rate for syringe one and syringe two. Then start the pump. After starting the pump, verify that there is no leaking from the swivel or from the cannulas and that the infusion tubing is not twisted.
Also, verify that there are no air bubbles in the tubing. After three hours of infusion, take a small blood sample and use a portable glucose monitor to measure glycemia drawing. Small volumes will prevent altering blood volume and or hematocrit.
Take additional blood samples using this timetable. Based on the measured glycemia values, modify the rate of syringe one to maintain blood glucose at 220 to 250 milligrams per deciliter. The rate of syringe two remains constant to maintain free fatty acids at one milli mole per liter.
After 24 hours of infusion, change the cage bottom and weigh the food remaining in the cage grill. Return the UNE portion to the cage, grill and refill syringes as needed throughout the 72 hours. Observe the rat for any signs of inflammation or discomfort and administer appropriate care as needed.
At the end of the infusion, rats can be subjected to hyperglycemic or u glycemic hyperinsulinemic clamp studies, and subsequently euthanized or euthanized to collect the pancreas for histology or eyelet isolation. This figure shows blood, glucose and fatty acid levels during the 72 hour infusion period. As shown here by design, the glucose levels are maintained around 220 to 250 milligrams per deciliter throughout the infusion.
Rodents have a strong capacity to compensate for the glucose infusion by increasing endogenous insulin secretion. Therefore, the GIR must be increased during the course of the infusion for blood glucose levels to be maintained at a relative steady state. Nevertheless, blood glucose levels tend to decrease towards the end of the infusion.
In addition, based on weight measurements of chow, since the glucose and intralipid infused animals are receiving caloric nutrients, they spontaneously decrease their food intake. Unlike the saline infused control rats once mastered, this technique can be performed in 30 to 40 minutes per rat if it is done properly. So following this procedure, we usually perform hyperglycemic or U glycemic hyper insulin anemic clamps to measure insulin secretion or insulin sensitivity respectively.
