The overall goal of this experiment is to investigate the role of hypothalamic C-C-L-5 signaling in insulin function in the peripheral system and glucose metabolism by manipulating C-C-L-5 signaling by administering the antagonist Met-C-C-L-5 through an intercerebral ventricular delivery system, the micro-osmotic pump. This medicine can help to answer the key questions in the biochemical physiology field, such as chemokine C-C-L-5 in controlling body glucose metabolism and insulin responsiveness in the brain. The main advantage of this measure is that the experimenter can easily manipulate certain signaling in the brain for a period of time, such as the C-C-L-5 in here, without compensatory effect or developing disorder, what usually happens when we do genetic manipulation in rodents.
Visual demonstration of this procedure is important as insertion of micro-osmotic pump and the accurate measurement of blood insulin and glucose level requires skill and precision. One day before the surgery, prepare brain micro-osmotic pumps by using a one milliliter syringe and blunt-head needle to fill the pumps with artificial cerebrospinal fluid, or A-C-S-F. Then, immerse the pump in A-C-S-F and place it on a shaker for gentle overnight shaking.
On the following day, remove the A-C-S-F from the pump and fill it completely with previously prepared drug solution diluted in A-C-S-F until the excess leaks out. Then, use surgical scissors to cut the catheter tubes to desired length. Using a blunt-end brain infusion needle attach the tubes to the brain infusion kit.
Then, fill the tubes in the infusion kit with the drug solution. After assembling the kit, attach it to the micro-osmotic pump. Finally, to prevent the pump from drying out, immerse the osmotic pump brain infusion setting in A-C-S-F within a sterilized 50 milliliter tube.
To start the surgery, mount and fix the head of an anesthetized mouse onto a stereotactic apparatus. Using a pair of surgical scissors and pincers cut open the outer skin covering the skull. Then, use iodine to clean the peripheral skull.
Next, use a pair of blunt-head scissors near the neck region to separate the outermost layer of skin from the subcutaneous skin for the osmotic pump brain fusion set implantation. Use the stereotactic apparatus to mark the infusion point with reference to the brain map. Use a nail drill to drill a hole around the area marked on the skull, being careful not to break the mouse meninges and blood vessels.
Then, place the micro-osmotic pump brain fusion set containing the drug of interest, or A-C-S-F as a control, under the skin behind the neck region. To infuse the drug into the brain, insert the brain infusion needle into the drilled hole. Use surface desensitizing gel to fix the needle in place on the skull and wait until the glue dries.
Cut off the projecting part on top of the needle. Before starting the glucose tolerance test, prepare the glucose solution by dissolving 3.75 grams glucose and 15 milliliters distilled water. Set up a timetable with proper intervals between each blood examination to record the readings during the experiment.
Next, weigh each mouse after fasting to calculate the appropriate amount of glucose for the injection. Then at the work bench, prepare a timer, glucose chip, glucometer, insulin syringe and razor blades. To measure the blood glucose level, first insert a new glucose chip into the glucometer and press the start button to set the zero.
Then, pick up the mouse at the back of the neck and stroke its tail to ensure sufficient blood flow. Use a new razor blade to cut off a small piece of the tail and then squeeze out a drop of blood into the glucose chip. Using intragastric lavage technique, feed the mouse glucose and immediately start the timer.
Measure the glucose at different time points. To start insulin tolerance test, prepare 0.25 units of human insulin solution. Then, weigh each mouse after fasting to calculate the appropriate amount of insulin for the injection.
Next, set up a timetable to record the readings during the experiment. Finally, after the insulin injection, measure the blood glucose level as previously done. Glucose metabolism was measured by the oral glucose tolerance test after the surgery, following the oral administration of glucose in mice.
The changes in blood glucose level in mice infused with A-C-S-F, or infused with C-C-L-5 antagonist, are recorded and shown here. The insulin sensitivity test was also performed and blood glucose level in mice infused with C-C-L-5 antagonist was only slightly reduced compared to control mice, suggesting impairments in insulin function on peripheral glucose metabolism in mice treated with C-C-L-5. To analyze insulin signal activation, insulin receptor substrate protein, or I-R-S-1 phosphorylation, was evaluated in hypothalamus.
In mice treated with C-C-L-5 antagonist when fed normally, I-R-S-1 phosphorylation was up-regulated compared to the control group. To further analyze insulin signal activation, levels of phosphorylated serine 4-7-3 Akt, insulin downstream signaling molecule, were also evaluated. After, insulin challenge A-K-T signal was increased in the control A-C-S-F treated mice but not in C-C-L-5 antagonist treated mice.
Once masters insertion of the micro-osmotic pump, will take just half an hour if done properly. While attempting this procedure it is important to ensure that the mouse head is slightly uplifted and the spine is straight on the stereotactic apparatus. This technique provides the researcher a way in you endocrinology to explore the effect of a drug, a chemokine, or a protein function in the rodent's brain.
Following this procedure, additional measure as manipulating gene expression with an intracerebral delivery of a virus, or crisper 9 can be performed in order to answer additional questions such as how does the C-C-L-5 and receptor C-C-L-5 participate in the food digestion and metabolism circuitry?
