The overall goal of this surgical intervention is to test the effect of therapies on amyloid beta oligomer toxicity in vivo. This method can help answer key questions in the field of Alzheimer's disease, such as finding new therapies targeting the neurotoxic effect of amyloid beta oligomer in an animal model. The main advantage of this technique is that it can be used to investigate mechanism by which amyloid beta oligomer induce neurotoxic effects in vivo.
And our treatment, like immunotherapy, may counteract the harmful effect on the brain. Demonstrating the procedure will be Chloe Provost, a technician from my laboratory. To begin the cannula implantation process, first cut PE50 catheters to six centimeters in length.
Then, fill the catheters with artificial cerebospinal fluid. Seal both ends of the catheters with a heat-seal machine. Add dental cement at both ends.
After anesthetizing rats according to an approved method, confirm anesthesia by checking movement after a gentle toe pinch. Then, inject the anesthetized animal with a non-steroidal anti-inflammatory drug. After the shaving the head of the animal with clippers, disinfect the skin with a solution of 2%chlorhexidine gluconate and 2%isopropyl alcohol.
Apply veterinary ophthalmic ointment on the eyes to prevent dryness while under anesthesia. Now, place the animal into a stereotaxic frame using the ear bars. Fix one cannula on the holder arm of the stereotaxic frame.
After injecting a local anesthetic agent and making a three centimeter scalpel incision on the top of the head, install four clamps around the incision to leave the skull clear. Next, use a round-tip scissor to make a two-by-two centimeter square pocket under the skin between the shoulder blades of the animal. Returning to the head, scratch the periosteum of the skull with a blade.
Apply a gauze pad on the skull if bleeding occurs. Now, to verify that the skull is flat and well-aligned on the stereotaxic frame, check the height coordinates at lambda and at bregma. Record the coordinates of bregma that will be used as the reference point.
After calculating the coordinates for correct bilateral placement of the two guide cannula, drill a 0.5 millimeter hole in the skull at the implantation point of both cannula. Drill three other approximately five millimeters above and below those points to insert the screws that will stabilize the cannula when applying dental cement. Next, cut one end of the catheter, previously filled with aCSF, with the scissor, and insert it in the angle arm of the cannula.
Fix the first cannula with dental cement. It is crucial to avoid putting dental cement around the position for the second cannula. After allowing the dental cement to dry for two to three minutes, remove the holder arm from the first cannula and place the trimmed second cannula in it.
Fix the second cannula in place with dental cement. Next, put dummy cannula on both guide cannula. Insert the free end of both catheters in the pocket previously made between the shoulder blades of the animal.
Remove the clamps, and then stitch the skin with a four-oh suture. Add more cement around the cannula. Remove the animal from the stereotaxic frame, and put it on a heat pad in a clean cage.
Monitor the animal constantly until it regains sufficient consciousness to maintain sternal recumbency. Then, return it to the housing room to recover from the surgery for 10 days under close monitoring. 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 two centimeters with a scalpel between the shoulder blades, locate the PE50 catheters connected to the guide cannula.
Next, cut the ends of the PE50 catheters containing dental cement with a scalpel. Connect the osmotic pumps to the PE50 catheters and add some dental cement at the pump and catheter junction to secure the connection. Finally, stitch the skin tightly with four-oh 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 one hour at room temperature before infusion. Then, fill each syringe with five microliters of sterile distilled water.
Next, cut two PE50 catheters to about 60 centimeters in length with a scalpel. Use two one-milliliter syringes fitted with 21-gauge needles to fill both catheters with sterile distilled water. While the one milliliter syringes remain in place at one end of each catheter, connect the free ends to Hamilton syringes.
Remove the one milliliter syringes and check that there are no air bubbles in the catheters. Insert the internal cannula at the end of the PE50 catheters. Use the Hamilton syringes to fill each internal cannula up to one microliter, then make an air bubble by pulling back the piston to the two 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 check point 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 one 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 micorliters, and that the air bubbles in both PE50 catheters move continuously. Check the rat to ensure that the catheters do not twist together.
Leave the internal cannula in place for another five 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 stop just before the angle arm of the guide cannula.
Finally, return the animal to its cage. The results presented here show the deposition of amyloid beta oligomers in the dentate gyrus close to the infusion site, and the cell death associated with this accumulation. The level of amyloid beta oligomers was substantially decreased by 6E10 antibody treatment as revealed by immuno-staining with anti-A-beta antibody.
Marked neuro-degeneration was observed near the injection site of A-beta-O and was attenuated by 6E10 antibody treatment as shown by Cresyl violet staining. Once mastered, cannula implantation can done in 30 minutes if it's performed properly. Osmotic pump installation will take about 10 minutes, and amyloid beta injection, about 15 minutes.
While attempting this procedure, it's important to remember to avoid putting dental cement around the position of the second cannula when inserting the first cannula. Also, wait until the animal calms down before inserting the internal cannula into the guide cannula. After watching this video, you should have a good understanding of how to implant cannula and osmotic pumps in order to infuse amyloid beta oligomers and test Alzheimer's disease treatments such as immunotherapy.
