Our research investigates how our brain regulates the homeo status of blood glucose levels and what kind of neurons, neuropeptides, and the neuron circuits do that. Additionally, how does our memory of food, psychological stress, pain, and other somatic sensations affected? It is difficult to assess glucose metabolism precisely.
We must check insulin sensitivity or resistance in insulin secretion and counter regulatory responses. We should also check which tissue changes glucose uptake and glucose production regarding insulin sensitivity or resistance. We believe that not only insulin sensitivity, but also insulin secretion and counter regulatory responses are all affecting normal blood glucose levels if we in healthy conditions.
We are trying to understand the whole picture of glucose regulation by the brain. Our protocol is low cost and does not need a special and expensive device to create a clamp. It will enable researchers to implement the use of the clamp easily and quickly.
Because this protocol helps researchers to use the clamp very easily, we expect our protocol will encourage more research in the entire field of life sciences. To begin, gather all the required sterilized tubings, polypropylene supplies and sutures. For assembling the jugular vein catheter or tubing 1.1, take eight centimeters of the polypropylene tubing one and glue one of its ends with three centimeters of the polyethylene tubing two.
Then place two millimeters of tubing one at the open end of tubing two. For the carotid artery catheter or tubing 1.3, stretch one end of tubing two to make it thinner, and then connect the other end with tubing one the same way as demonstrated previously. Make a mark at nine millimeters from the thinner end or the tip.
For the surgery, fill heparinized saline in the assembled jugular vein and carotid artery tubings, and connect each to a one milliliter syringe fitted to a non-sharp needle. Close the end of the tubings with caps made by soldering one end of tubing three. To begin the surgery, keep the properly anesthetized mouse on the warm pad at 37 degrees Celsius to reduce physical stress.
Wipe the two shaved incision sites three times each with 10%Betadine, followed by sterile 70%alcohol. After making a vertical incision five millimeters cephalic to the sternum, expose the right carotid artery while separating the vagus nerve from it. Stop the blood flow by placing two silk sutures under the artery and the other loosely on the coddle side, and tying one tightly on the cranial side.
Place one more suture under the artery. Using spring scissors, cut the artery near the cranial suture to place the carotid artery catheter into it, and loosely tie both the artery and the tubing. With the coddle side knot open, insert the tube until the nine millimeter mark reaches the knot in the middle.
After securely tying all ligatures, flush the tubing with heparinized sterile saline. Next, expose the right jugular vein from the same incision as the right carotid artery. Ligate the isolated cranial end with silk suture, and then place the suture at the coddle end of the exposed vein.
Using spring scissors, cut the vein near the cranial suture. Insert the jugular vein catheter fitted to the syringe into the vein and tie it. Before visually confirming that it samples blood, flush with 0.2 milliliters of heparinized sterile saline and visually confirm that no blood remains in the catheter.
After wiping the second incision site on the back as demonstrated previously, make a small incision between the shoulder blades. Pass a needle holder under the skin from the incision on the back of the ventral side. Clamp the implanted catheters to the needle holder, pass them under the skin and bring them back.
After cleaning the incision site, close the ventral incisions with a synthetic suture. Then, suture the dorsal incision. Clamp the venous catheter with micro serrefine at the incision site between the shoulder blades.
Cut the catheter one centimeter above the clamp and flush it with heparinized saline before closing it with a cap. Clean the skin around the incision after surgery with an alcohol prep pad. Follow proper postoperative care and monitoring.
Begin by setting up the pump system for the hypoglycemic or hyperglycemic clamps. To do so, fill two hamilton syringes with the insulin infusion and 30%glucose solution. Connect the filled syringes to the two long tubings of the extension tube setup meant for glucose and insulin infusion.
After attaching the syringe assembly to the respective syringe pumps, fill each solution into the respective extension tubings. Next, fill saline in the one milliliter syringe and the extension tube for drawing blood from the artery. Next, connect the extension tube for infusion to the pre-installed jugular vein catheter of the properly anesthetized mouse, the extension tube for drawing blood to the carotid artery catheter of the animal.
Use cellophane tape to secure the tubes together. Place the mouse with the tubings inside an empty 500 milliliter beaker with bedding. Clamp the top end of the extension tube for drawing blood while connecting it to a new syringe.
Extract 50 microliters of blood, transfer it to a 1.5 milliliter tube, and measure the blood glucose level. Then, infuse 50 microliters of saline. To collect blood for hormone measurements, extract an additional 50 microliters of blood and transfer it to a 1.5 milliliter tube on ice.
After the zero minute blood glucose measurement, start the insulin syringe pump to infuse the appropriate amount of insulin. Keep measuring the blood glucose level and change the glucose infusion rate every five to 10 minutes during the 120 minute monitoring time until a steady state is reached. For the hyperglycemic clamp, follow the steps demonstrated for the hypoglycemic clamp, excluding insulin infusion, and an adjusted steady state blood glucose level.
