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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of hemoglobin and CSF solutions
- Prepare a sterile artificial cerebrospinal fluid (aCSF) solution by adding 500 µL of the aCSF solution to a 1.5 mL microtube and store on ice.
- Prepare a sterile 150 mg/mL hemoglobin solution by adding 75 mg of hemoglobin to 500 µL of aCSF in a 1.5 mL microtube and store on ice.
2. Preparation of the animal for injection
- Turn the heating pad to the medium setting to maintain the body temperature of the rat.
- Anesthetize postnatal day 4 (P4) rats in an induction chamber filled with 3% isoflurane.
NOTE: Confirm sufficient anesthesia using the toe/tail-pinch response every 15 min. Monitor anesthesia with visual observation of tissue color, body temperature, and respiratory rate.
- Administer pain relief with a 5 mg/kg subcutaneous carprofen injection to the anesthetized rat.
- Place the anesthetized rat prone in the stereotactic apparatus with the nose positioned in the anesthesia adaptor with a constant flow of 1.5% isoflurane.
- Tighten non-rupture ear bars on the external auditory meatus to secure the head.
NOTE: Apply vet ointment to keep the eyes moisturized if the eyes are open at the age of injection.
- Clean the head, alternating with sterile cotton-tipped applicators soaked in betadine and 70% ethanol.
- Touch the betadine-soaked applicator to the center of the scalp and spread the betadine in circles, moving outward.
- Repeat step 2.6.1.1 with the ethanol-soaked applicator.
- Repeat step 2.6.1.1 and step 2.6.1.2 3x.
- Apply a sterile surgical drape to protect the surgical field.
- Using a sterile scalpel, make a 0.3 cm incision vertically down the center of the head to expose the bregma of the skull.
NOTE: If injecting from the lambda, expose the lambda of the skull instead of the bregma.
- Use a sterile cotton-tipped applicator to dry the area.
3. Setting up the stereotactic injector
- Draw the hemoglobin solution prepared in step 1.2 into a 0.3 mL sterile syringe with a 30G needle and place the syringe into the stereotactic injector system.
NOTE: If generating control conditions, draw aCSF solution prepared in step 1.1 into a 0.3 mL sterile syringe and proceed with the protocol.
- Turn on the stereotactic injector interface and click on the Configuration button to input the injection volume and rate settings.
- Click on Volume and set the volume at 20,000 nL (20 µL).
- Click on Infusion rate and set the rate at 8,000 nL/min (8 µL/min).
- Exit Configuration by clicking on the Reset Pos button.
- Flush the needle tip by clicking on the Infuse button until a small bead of hemoglobin solution emerges at the needle tip.
- Gently wick the hemoglobin solution from the needle tip with a sterile cotton-tipped applicator.
4. Animal injection
- Set the bregma as zero on the stereotactic injector system by adjusting the mediolateral and anteroposterior positions of the syringe before lowering the tip of the flushed syringe needle to gently touch the skull at the bregma.
NOTE: If injecting from the lambda, set lambda as zero.
- Identify the coordinates of choice.
- If injecting from the bregma, in P4 rats described here, use 1.5 mm lateral, 0.4 mm anterior, and 2.0 mm deep from bregma.
- If injecting from the lambda, use the following coordinates for P4 rats: 1.1 mm lateral, 4.6 mm anterior, and 3.3 mm deep from lambda.
- Raise the syringe needle 1 cm above the skull to clear the scalp. When the syringe is raised, proceed to set the mediolateral and anteroposterior coordinates.
- Lower the syringe needle to gently touch the skull. Check that the needle is touching the skull.
- Set the dorsoventral coordinate over a 30 s period.
NOTE: While setting the dorsoventral coordinate, the needle will puncture the skull. Care must be taken to assure that the syringe passes through the skull without deforming the skull. Skull deformation is avoided by slowly withdrawing the needle along the dorsoventral coordinate if deformation occurs, then placing the needle back along the same trajectory. This allows the needle to pass through the hole in the skull with less force and no deformation.
- On the stereotactic injector interface, click on the Run button to begin injection.
- After the injection is finished, leave the syringe needle in place for 2 min to minimize backflow of the solution.
- Withdraw the syringe slowly along the dorsoventral coordinate over 2 min until the needle tip is 2 cm above the scalp.
- Rotate the stereotactic injector arm away from the operative field.
5. Postoperative care
- Close the scalp with a 6-0 monofilament suture. Make one simple interrupted suture at the center of the 0.3 cm incision.
- Remove the pup from anesthesia and place it onto a secure area on the heating pad.
- Return the rodent to the home cage to recover from the anesthesia under the care of its dam.
NOTE: Timely return to the care of the dam reduces early postoperative mortality.
- Monitor the animals for anesthesia by loss of the righting reflex hourly post-surgery for 3 h.
- Monitor the animals daily for 7 days for normal activity, food intake, and weight gain. Monitor the incision site for wound healing, closure, and reappearance of fur at the site of surgery.
NOTE: In the rare case that neurological changes such as seizures, central depression, or decreased appetite are observed during monitoring, euthanize the animal using intravascular perfusion or cervical dislocation under anesthesia.
- To prevent infection once the suture is closed and the wound is healing, apply topical triple-antibiotic at the incision site.