Controlled Infusion of Aβ Peptide-Specific Antibodies and Aβ Oligomers into the Rat Hippocampus
Transcrição
One day before installation, fill the osmotic pumps with 6E10 antibody or control IgG1 antibody according to the manufacturer's instructions. Place the pumps in sterile distilled water and keep at 37 degrees Celsius overnight to activate the pumps.
Once the rat has been anesthetized with 3% isoflurane and shaved between the shoulder blades, disinfect the skin with a solution of 2% chlorhexidine gluconate and 2% isopropyl alcohol. After making an incision of 2 centimeters with a scalpel between the shoulder blades, locate the PE-50 catheters connected to the guide cannula.
Next, cut the ends of the PE-50 catheters containing dental cement with a scalpel. Connect the osmotic pumps to the PE-50 catheters and add some dental cement at the pump and catheter junction to secure the connection. Finally, stitch the skin tightly with 4/0 sutures. Put the animal back in its cage on a warm heat pad, and monitor it as it awakens from isoflurane anesthesia.
After preparing the amyloid-beta oligomer solution as previously reported, allow the solution to aggregate dynamically and spontaneously for 1 hour at room temperature before infusion. Then, fill each syringe with 5 microliters of sterile distilled water.
Next, cut two PE-50 catheters to about 60 centimeters in length with a scalpel. Use two 1-milliliter syringes fitted with 21-gauge needles to fill both catheters with sterile distilled water. While the 1-milliliter syringes remain in place, at one end of each catheter, connect the free ends to Hamilton syringes. Remove the 1-milliliter syringes and check that there are no air bubbles in the catheters.
Insert the internal cannula at the end of the PE-50 catheters. Use the Hamilton syringes to fill each internal cannula up to 1 microliter, then, make an air bubble by pulling back the piston to the 2 microliter mark.
Now mix the amyloid-beta oligomer solution by pipetting up and down using a tip of minimum adherence. Avoid forming bubbles during mixing. Then, fill both internal cannulas with 1.5 microliters of amyloid-beta oligomer solution by pulling the pistons back to 3.5 microliters.
Next, mark lines before and after the air bubble in both catheters. This serves as a checkpoint of ongoing infusion. Place the awake rat in a snuggle hold and immobilize its head. Remove the dummy cannula from the two-guide cannula, then insert the previously prepared internal cannula into the guide cannula. Verify that they are fully inserted and well-fixed to the base of the guide cannula. Now, release the rat from the snuggle hold, and put it back in its cage to limit handling stress.
Turn on the infusion pump and inject 1 microliter of amyloid-beta oligomer solution at a rate of 0.1 microliters per minute. During infusion, check that the syringe piston moves from 3.5 microliters to 2.5 microliters, and that the air bubbles in both PE-50 catheters move continuously. Check the rat to ensure that the catheters do not twist together.
Leave the internal cannula in place for another 5 minutes after infusion to allow efficient diffusion of amyloid-beta oligomer solution. After infusion, bring the rat back in the snuggle hold, and remove the internal cannula and capped-guide cannula to prevent reflux of the injected solution. Place dummy cannula that stopped just before the angle arm of the guide cannula. Finally, return the animal to its cage.
