$$\rightleftharpoonup{xx}$$
$$\longleftharp{xx}$$,
$$\longrightharp{xx}$$,
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Micro-Osmotic Pump Infusion Systems
NOTE: Prepare the pump, artificial cerebrospinal fluid (aCSF) buffer, and drug (Met-CCL5/RANTES protein solution (10 ng/mL in aCSF)) under sterile conditions using buffers filtered with 0.2 µm filters and conduct all the procedures under the culture hood with gloves. The surgery procedures are conducted as follows:
- Prepare the micro-osmotic pumps one day before surgery: Fill the brain micro-osmotic pump with artificial cerebrospinal fluid (aCSF) with a 1 mL syringe and blunt-head needle provided along with the kit. Immerse the micro-osmotic pump in aCSF and place it on a shaker with gentle shaking overnight.
CAUTION: The pump should be filled with aCSF, and air bubbles should be avoided inside the pump (Figure 1A).
- Before starting surgery, prepare the recombinant Met-CCL5/RANTES protein solution (10 ng/mL, diluted in aCSF) for the experiment. Remove aCSF from the pump and fill the pump with the drug solution slowly until excess leaks out.
NOTE: 15 mL aCSF or Met-CCL5/RANTES solution is sufficient for 5-8 pumps.
CAUTION: Repeat the procedure to ensure that the pump is completely filled with the drug without bubbles inside.
- Cut the catheter tubes into the desired length and attach them to the brain infusion kit's blunt-end brain infusion needle. Fill the infusion kit and tubes with the drug.
- Finally, assemble and attach the brain infusion kit to the micro-osmotic pump.
CAUTION: No bubbles should be formed in the tube or the pump (Figure 1A).
- Immerse the entire osmotic pump-brain fusion set in aCSF in a sterilized 50 mL tube to prevent the pump from drying out. The osmotic pump-brain fusion set is now ready to be used for surgery.
NOTE: The micro-osmotic pump systems can be used for long-term drug infusion. This ensures a safe and convenient mode of drug delivery into the mouse brain.
2. Intracerebral Ventricular Surgery – implantation of the micro-osmotic pump
CAUTION: Sterilize the surgical environment with 75% ethanol and ensure that the people involved in the study are wearing sterile gloves and a clean lab coat. Surgical tools/ instruments must be autoclaved and dried before use and subsequently sterilized with 75% ethanol in-between mice surgeries.
- Weigh the mouse and anesthetize it using intra-peritoneal injection (IP) with Ketamine/Xylazine (Ketamine 50 mg/kg, Xylazine 10 mg/kg).
CAUTION: Mice body weights lower than 24 g are not recommended for osmotic pump implantation surgery.
- Mount and fix the mouse head onto the stereotactic apparatus (Figure 1B).
- Use a pair of surgical scissors and pincers to cut open the outer skin covering the skull. Use iodine to clean the peripheral skull.
- Separate the outermost layer of skin from the subcutaneous skin with the help of a pair of blunt-head pincers near the neck region for the osmotic pump-brain fusion set implantation (Figure 1C).
- Mark the infusion point with reference to the brain map (Figure 1D) using the stereotactic apparatus. In this experiment, the needle needs to be implanted in the 3rd ventricle region (Bregma: 0.0 mm lateral, 1.3 mm posterior, 5.7 mm ventral).
- Drill a hole using a nail drill around the area marked on the skull (Figure 1E).
CAUTION: Be careful not to break the mouse meninges and blood vessels, thus avoiding the disruption of micro-blood vessels in the brain.
- Place the micro-osmotic pump-brain fusion set containing aCSF (as control) or drug (Met-CCL5/RANTES protein solution) under the skin behind the neck region and insert the brain infusion needle into the drilled hole to infuse the drug into the mouse brain (Figure 1E). The needle will penetrate the meninges and get into the ventricle. Fix the needle in place on the skull using surface desensitizing gel (Figure 1F) and wait 1-2 min until the glue dries. Next, cut off the projecting part on top of the needle (Figure 1G-H).
- Use a tissue adhesive glue to heal the operation wound on the head. Apply 50 µL of the glue on top of the wound, pull both sides of the skin together, and hold on for 30 s to allow the skin to seal (Figure 1I).
CAUTION: Use a 100% alcohol pad to clean the wound after surgery and 100 ul penicillin with streptomycin to prevent infection. NOTE: Mouse skin will form scar tissue and heal in a few days following the administration of the surgical glue. The main advantage of the glue is the avoidance of surgical stitches, which may cause skin irritation or inflammation.
- Place the mouse in a clean cage kept on a warm plate (heated up to 37 °C) and wait until the mouse recovers from the anesthetic effect.
CAUTION: It is critical to maintain the mouse's body temperature to enhance the chance of survival after surgery.
- After a one-week recovery period, the mice will be ready for further experiments, such as the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT).
3. Oral Glucose Tolerance Test (OGTT)
NOTE: Perform the oral glucose tolerance test 7 days after the infusion of aCSF and MetCCL5/RANTES (10 ng/mL, 100 µL). Maintain a 6 h fast for the mice before OGTT with sufficient water supply. Keep the animals on the same work bench where the experiments will be performed so that they can acclimatize to the environment to reduce stress during the procedure.
- Glucose solution preparation: Before conducting the experiment, prepare the glucose solution by dissolving 3.75 g glucose in 15 mL of distilled H2O.
- Set up a timetable to record the readings during the experimental procedure (Table 1).
NOTE: It is important to set up a timetable with proper intervals between each blood examination to allow accurate recording during the experiment.
- Weigh each mouse after fasting and calculate the appropriate amount of glucose to be injected. For example, if the mouse weighs 30 g, the amount of glucose solution to be administered should be 300 µL.
- Prepare the following instruments at the workbench:
- Glucometer (Press the start button to check the battery status; make sure it is functioning before the test.)
- Glucose chip
- Insulin syringe (0.3 mL Insulin syringe)
- Razor blades
- Timer
- Once the bench is set up, measure and record the blood glucose level as follows: Put a clean and new glucose chip into the glucometer and press the start button to zero it.
- Pick up the mouse by the back of the neck and stroke the tail a few times to ensure sufficient blood flow to the tail region.
- Using a new razor blade to cut off a small piece of the tail and squeeze out one small drop of blood (about 10-20 µL) into the glucose chip. The blood should fill the chip to allow accurate measurement. The glucometer will display the glucose level immediately. If the machine shows an "error," repeat the procedure with a new glucose chip.
NOTE: The glucose chip requires only one drop of blood. When the blood sample needs to be collected more than once, simply apply pressure by running your fingers along the mouse's tail several times while holding the end of the tail directly on top of the chip to collect blood. It is not necessary to cut the tail end each time while collecting blood samples.
- Next, feed the mice glucose (0.25 g/mL) orally by using the intragastric gavage technique. The amount of glucose to be administered should be calculated by using the formula: 10X body weight (BW) µL glucose solution (for example, if the mouse weighs 30 g, the amount of glucose solution to be administered will be 300 µL). Start the timer immediately after administering oral glucose.
- Repeat the glucose measurement procedure at 15, 30, 60, 90, and 120 min.
- After all the glucose level readings have been recorded, discard the razor blades and glucose chips in a biohazard container. Put the food back into the mice cages and return them to the animal room.
Table 1. Timetable for Oral Glucose Tolerance Test (OGTT) recording
| # | Mouse ID | Body | Glucose | Start | 0 | 15 | 30 | 60 | 90 |
| | weight | μL =10xBW | time | mins | mins | mins | mins | mins |
| 1 | 501 | 25.8g | 258 | 9:00 | 9:00 | 9:00 | 9:15 | 9:30 | 10:00 |
| 2 | 502 | 25.3g | 253 | 9:07 | 9:07 | 9:07 | 9:22 | 9:37 | 10:07 |
