The overall goal of this procedure is to evaluate insulin sensitivity in the conscious unrestrained rat. This is accomplished by first catheterizing, the left carotid artery. Next, the right jugular vein is catheterized to ensure optimal results.
The catheters are flushed daily to maintain patency during a five day postoperative recovery period. Ultimately, results can be obtained from the hyperinsulinemic U glycemic clamp that show whole body glucose infusion rates. The main advantage of this technique is that direct measurements of whole body glucose disposal rates can be obtained at high physiological insulin levels.
Arterial and venous catheters must be prepared in advance of the surgical procedure. The catheter should have blunt ends be filled with heparinized saline and be ethanol sterilized with a restraining beat securely attached to 2.7 or 3.2 centimeters from one end for arterial and venous catheters respectively. Adult male spray dolly rats of approximately 300 grams are used in this procedure.
Record the starting weight of the animal and use this weight to monitor post-surgery weight gain. Working in an aseptic surgical area. Anesthetize the rat with 3%isof fluorine by placing it into an anesthetic box.
Transfer the animal away from the surgical area and prepare the incision site by removing hair from around the neck and shoulders. Once the hair is removed, place the animal in a nose cone to maintain anesthesia with approximately 2%isof fluorine. Secure the animal to the surgical table.
Ensure that the rat is fully anesthetized by checking for the presence of foot and or eye reflexes. Clean the incision site with 70%ethanol, followed by a Betadine scrub and followed again by 70%ethanol. The first step of the procedure is to make a small vertical midline incision 10 millimeter superior to the sternum.
Use curved micro dissecting forceps to blunt dissect tissue away and expose the left sternal mastoid muscle. Reflect this muscle to reveal approximately one centimeter of the left carotid artery. Place micro dissecting forceps under the carotid artery to hold it in place while gently teasing away connective tissue.
It is important to isolate the vagus nerve away from the artery without damaging either the artery or the nerve. Use sterile four oh silk suture to ligate the cephalad end. Clamp the vessel with serrated micro dissecting forceps.
Puncture the ligated. End with a 21 gauge ven eject multis sample lure adapter. Remove the stainless steel plug from the catheter and use a sterile catheter introducer to guide into the artery.
Use forceps to ensure the catheter is secure.Partially. Release the micro dissecting forceps, clamping the artery and continue to insert the catheter up to the bead. At this point, the tip of the catheter should be in the aortic arch.
Secure two sutures below the bead and one above, and flush the line with 10 units per milliliter heparinized saline. To confirm that the catheter will sample replug the external end of the catheter with a stainless steel plug. Next blunt dissect tissue To expose the right external jugular vein, isolate the vein carefully and ligate the cephalic.
End with silk suture. Puncture the vein with a 21 gauge vein. Eject multis sample lure adapter.
Remove the stainless steel plug from the catheter and use a sterile catheter introducer to insert it up to the bead. Secure two sutures below the bead and one above. And flush the line with 10 units per milliliter heparin ice saline.
To confirm that the catheter will sample replug the external end of the catheter with a stainless steel plug using a 14 gauge blunt needle tunnel under the skin and make an incision in the back between the shoulder blades. Next, thread the catheters through the needle so that they exit from the back of the rat. Approximately four centimeters of each catheter should be visible.
Cut 0.5 centimeters of Tigon S 50 HL medical tubing and place it around both the exteriorized catheters. Secure with blue tape for veins and red for arteries. Retest catheters to ensure patency and then flush and fill with 150 units per milliliter heparinized saline to prevent clotting.
Close all incisions with three oh silk suture. Place the rat in the prone position in a pre-warned clean cage with food easily accessible. Once the rat regains full ambulatory ability and alertness, return it to the home cage.
Allow the rat to recover for three to five days monitoring daily for infection, pain, and changes in weight. It is important that catheter patency is maintained throughout the study. Each day following surgery, fill one milliliter slip tip syringes with 150 units per milliliter heparinized saline and cap them with a 22 gauge blunt needle.
The blunt needle is inserted into 20 centimeters of PE 50 tubing with 23 gauge stainless steel tubing coupler at the other end. Be sure to remove all air bubbles from the line Clamp off the arterial line externalized from the rat with hemostats. Just below the plug.
Remove the steel catheter plug using another pair of hemostats. Insert the tubing coupler connected to the syringe into the arterial line and release the hemostats occluding the externalized arterial catheter. Next, aspirate the arterial blood of the catheter into the syringe.
If the catheter does not draw easily, it may be necessary to very gently push in a small amount of solution through the catheter to dislodge the tip. The catheter is considered cleared. When blood reaches the syringe clamp the P 50 tubing close to the one milliliter syringe and dispose of the syringe.
Replace with a new syringe filled with fresh heparin ice saline. Un clamp the tubing and while holding the new syringe upright, flick the new syringe to dislodge air bubbles upwards and away from the line. Inject heparinized saline until the catheter line is clear and free of blood.
Clamp off the arterial line just below the tubing coupler. Remove the syringe connector from the arterial catheter line and replace it with the stainless steel tubing plug. Release the hemostats occluding, the externalized arterial catheter.
Repeat this procedure for the venous line. Due to low venous pressure sampling may not be possible. In general, if the heparinized saline can be infused with minimal resistance, it is likely that the catheter is well positioned in the vein.
Before beginning the procedure, animals must be weighed solutions prepared and the clamp apparatus assembled After the animal is weighed and catheter patency confirmed, place it in a small container with bedding that limits but does not restrict movement. Rats used in this study are fasted for at least five hours and have met postoperative weight gain requirements. The weight of the rat determines the amount of insulin required here.
Four milli units per kilogram per minute is administered at a rate of two microliters per minute. Prepare a 50%dextrose solution and be sure that there is enough of both solutions to last around two hours here, three milliliter syringes are used. Now you are ready to assemble the clamp apparatus.
Set up the lines and infusion pumps as seen in here. Place glucose and insulin syringes onto the syringe pump. Clamp the lines and allow the rat to relax for 30 minutes prior to starting the experiment.
To begin the procedure, take a baseline insulin and hematocrit sample hematocrit sampling ensures blood volume is maintained throughout the experiment. Generally, hematocrit levels should not fall more than 10%of the initial value. Obtain a baseline glucose sample and determine glucose level to be clamped.
Here we clamp at U glycemia or five millimolar. After each blood sample, flush the arterial line with a small volume of 10 units per milliliter heparinized saline to prevent clotting. After determining the glucose level to be clamped, start the infusion of insulin solution.
Record blood glucose at five to 10 minute intervals and adjust the glucose infusion rate as required until a steady state is achieved. This is generally a trial and error process and can take anywhere from 30 minutes to more than two hours. The level of glucose required to maintain glycemia is dependent on the experimental protocol.
Species and conditions present in order to be considered clamped, three consecutive readings must fall within a defined range. Three readings within approximately one millimolar of each other implies that glucose levels have reached a steady state record. The glucose infusion rate required to maintain glycemia for a 30 minute period, additional blood samples can be obtained during this time at minimum.
A second plasma insulin hematocrit sample should be collected once the clamp procedure is complete. Animals can be used for further testing or euthanized in tissues collected for further analysis. Depending on the animal catheter lines can stay patent for five to seven days when performed correctly.
The clamp procedure can measure the steady state insulin sensitivity. It is essential to record insulin and glucose levels as well as glucose infusion rates and hematocrit values. Steady state is reached when glucose levels remain within one millimolar of each other over a 30 minute time period.
The glucose infusion rate reveals the level of exogenous glucose required to maintain U glycemia where possible rate should be grafted over time as opposed to a single average time point. Both the fasting and clamped insulin levels should be reported. These measures confirm that insulin was delivered successfully and will detect any differences in the insulin response between treatment groups measuring hematocrit levels at baseline, and again after the procedure assures that levels have not fallen more than 5%of baseline over the course of the experiment Following this procedure.
Other methods such as isotopic tracer administration may be used to evaluate whole body and tissue specific glucose utilization.
