Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

The overall goal of this procedure is to stereotaxic inject a Liga meric a beta 1 42, into the dentate gyrus of the hippocampus. This is accomplished by first preparing a liga meric, a beta 1 42 solution. The second step is to determine the injection coordinates.

Next surgically, expose the skull of the animal and introduce two holes in it for needle insertion. The final step is to move the syringe needle into its final position and to infuse a beta 1 42. Ultimately, the Stereotaxic Abeta 1 42 injection method is used to examine the toxic effects of Abeta 1 42 in a live animal.

The main advantage of this technique over existing transgenic, Abeta 42 mouse models is that oligomeric ALA 42 can be elevated in the special and temporal manner and reliably reproduce the cell death pathology that is lacking in the transgenic mouse models. This method can help answer key questions in the Alzheimer's field, such as how pathological processes spread in Alzheimer's disease. To begin this procedure, shave the head of an anesthetized mouse to expose the skin.

Next, place the mouse on a heating pad in a stereotaxic frame. Then secure its incisors in the teeth guard and a nose piece over the face of the mouse. After that, position the ear bars into the auditory mauss in order to secure the head to check.

If the head is secured, gently press it and ensure that it doesn't move. Then apply ointment to both eyes to keep the moist. After that, disinfect the surgical site by applying Betadine to the skin over the skull, followed by 70%ethanol with sterile cotton swabs.

Perform the alternating Betadine and ethanol cleaning two more times. Now make a two to three 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 bgma 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 Bre ma position the Hamilton syringe using the micro manipulator so that the tip of the needle just touches the dot on the bgma. 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 bgma point. After that, record the DV position. The DV coordinates for Bre, MA and Lambda should be within 0.5 millimeters.

Now return the needle tip to the BMA dot. Record the starting AP position. Then calculate the ending AP position by subtracting two 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 one 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 db.

Coordinate then set the stereotaxic injector pump to infuse four microliters of a beta 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 the suture. Take off the nose guard and remove the mouse.

Inject one milliliter of sterile saline into the mouse intitally for hydration and inject buprenorphine at 0.1 milligrams per kilogram subcutaneously for pain relief. Afterward, transfer the mouse to a clean empty cage set on top of a 42 degree Celsius hot plate until it wakes up. Then return the mouse to its housing cage with ample food and water.

This figure shows the infusion of a beta 1 42 and vehicle in the respective left and right dentate gyrus of a nine month old mouse. Broken lines depict the injection tracted and the arrows show the collapsed dentate gyrus. Within one week of Abeta 1 42 injection, the dentate gyrus exhibited elevated Abeta 1 42 levels in the molecular layer and the polymorphic cell layer close to the injection site at seven and 14 days.

Abeta 1 42 elicited increases in tunnel positive staining and decreases in dentate gyrus thickness, confirming the neurodegenerative effects of Abeta 1 42. Here, a retrograde tracer fluoro gold was co injected with Abeta 1 42 into the dentate gyrus. Seven days following injection, fluorgold was detected in the basal forebrain suggesting the retrograde transport of the label via cholinergic neurons.

Fluorgold positive neurons in the basal forebrain were chosen for quantification. Seven days following injection, Abeta 1 42 elicited increases in tunnel and decreases in chat. Positive neurons in the basal four brain Once mastered this technique can be done in approximately 30 minutes if performed properly.

Following this procedure, other methods can be used to answer additional questions such as studies of molecular mechanism to address the mechanistic effect of amyloid infusion on the brain. I'd like to thank you for watching our video and I hope that the method will be useful in your experiments.