Direct Stereotaxic Brain Infusion of Amyloid-Beta Proteins for an Alzheimer’s Disease Model

Begin with an anesthetized mouse with an exposed skull. The mouse is secured in a stereotaxic frame containing a syringe pre-filled with aggregation-prone amyloid-beta proteins.

Mark the bregma, a key reference point for determining protein infusion coordinates.

Move the syringe, and touch the bregma, then the lambda, to determine the dorsal-ventral coordinates, ensuring precise needle depth.

Reposition the syringe above the predetermined medial-lateral coordinate, targeting the dentate gyrus, crucial for memory processing.

Touch the skull surface, ensuring the dorsal-ventral coordinate is within the limit, and mark it. Repeat this procedure on the opposite side.

Drill at the marked medial-lateral coordinate, inject the amyloid-beta proteins into the dentate gyrus, and hold to prevent backflow.

Drill and inject amyloid-beta proteins on the opposite side for uniform protein dispersion.

Detach the ear bars from the mouse, then suture the incision.

Over time, amyloid-beta proteins aggregate to form plaques that degrade dentate gyrus neurons and disrupt memory processing, resulting in Alzheimer's disease.

Make a 2- to 3-millimeter incision in the midline of the scalp with a scalpel. Use straight fine scissors to extend the incision line to about 1.0 centimeters to expose the sagittal suture, bregma, and lambda of the skull. After that, use forceps to pull the skin apart.

Clean the skull with a sterile cotton swab soaked with sterile saline. Then use a clean, dry cotton swab to dry the skull. Next, mark a dot on the bregma. Position the Hamilton syringe using the micromanipulator so that the tip of the needle just touches the dot on the bregma. Then, record the DV coordinate.

Mark a dot on the lambda point. Make sure that the AP axis of the skull is level by moving the syringe posteriorly to the lambda point. The tip of the needle should touch the lambda point as it did the bregma point. After that, record the DV position. The DV coordinates for bregma and lambda should be within 0.5 millimeters.

Now, return the needle tip to the bregma dot. Record the starting AP position. Then, calculate the ending AP position by subtracting 2 millimeters from the starting AP coordinate, and reposition the syringe needle to the final AP coordinate.

Next, record the current ML coordinate. Calculate the left and right ending ML coordinates by adding or subtracting 1.3 millimeters from the starting ML coordinate, and move the syringe needle to the ending ML coordinates. Touch the needle tip to the surface of the skull on both ending ML coordinates, and make sure the DV coordinates are within 0.5 millimeters.

While at the ending ML coordinate, pull the needle up 0.5 to 1 centimeters over the skull. Mark a dot on the skull with a dark-colored extra-fine-point marker. Repeat this step for the contralateral side of the skull. Then, move the syringe away.

Drill a hole on either the left or right ML coordinate on the skull using a 0.8-millimeter drill bit. Repeat this step for the contralateral side. Afterward, move the syringe to one of the newly introduced holes. Lower the needle to just past the skull, and be careful not to puncture the brain.

To ensure that the needle has passed the skull, gently push the needle against the skull to make sure that the needle doesn't come out of the hole. Next, record the starting DV coordinate. Calculate the ending DV coordinate by subtracting 2.2 millimeters from the starting DV coordinate, and lower the needle to the ending DV coordinate.

Then, set the stereotaxic injector pump to infuse 4 microliters of Aβ_1-42 into the dentate gyrus at a rate of 0.5 microliters per minute. After the infusion, let the needle remain in place for an additional minute to minimize backflow of solution out of the injection site. After that, move the needle to the other side of the brain and repeat the procedure.

Now, unscrew the ear bars. Use student standard pattern forceps to pull the scalp closed and seal the wound with a suture. Take off the nose guard and remove the mouse.