Step 1. Determine the correct stereotaxic coordinates for the target infusion site and calculate the desired cannula length.
Step 1. Stereotaxic data for placement of cannulae are available in: Paxinos G and Watson C. The Rat Brain in Stereotaxic Coordinates. Academic Press, San Diego, 1997.
Step 2. Without modification, the L-shaped cannula included in the ALZET Brain Infusion Kits will penetrate approximately 5 mm below the surface of the skull when using the Brain Infusion Kit 1 or 2 and 3 mm for the Brain Infusion Kit 3. Depending on the animal size, skull thickness, and desired site for infusion, this depth may need to be altered. Spacers are included in the ALZET Brain Infusion Kits to allow you to alter the cannula depth. To do so, attach the desired number of 0.5 mm depth adjustment spacers to the bottom of the cannula with cyanoacrylate adhesive.
Step 3. After attachment of the spacers, verify the length of the remaining cannula tube. Also verify that the tube is straight and that it is at right angles to the bottom of the cannula and spacers. Should the cannula become contaminated during this procedure, soak it in an aqueous solution of 70% ethanol for several minutes. Before implantation, allow the ethanol to evaporate from the surface of the cannula and from the interior of the cannula tube.
Step 4. Ten 15 cm lengths of catheter tubing are included in the Brain Infusion Kits. This tubing can be used to attach the cannula to the flow moderator of the ALZET pump. First, measure the distance between the location at which the cannula will be placed and the site of pump implantation. The catheter which connects the cannula to the pump should be 25% longer than the distance between the subcutaneous site of the pump and the location of the cannula, to allow free movement of the animal’s head and neck. For intraventricular infusion in a rat weighing 250 grams, 10 cm of tubing is typically required.
Step 2. Note: Attachment of more than four spacers to each cannula is not recommended as this may compromise stability or cause the top of the cannula to protrude too far above the skull. This may make it difficult to close the scalp incision after cannula placement.
Step 5. Cut the catheter tubing to the length determined in Step 4. Attach one end of the tubing to the cannula and the other end to the ALZET pump flow moderator. Check the attachment by gently pulling on the catheter. Neither end of this assembly should be loose or easily dislodged. If you are concerned about the security of this assembly, cement the catheter to the cannula and the catheter to the flow moderator with cyanoacrylate adhesive. The brain infusion assembly is now complete.
Step 5. Note for attaching catheter to the pump:
200 µl & 2 ml Models: Remove the translucent cap from the end of the flow moderator, revealing a short stainless steel tube protruding from the white flange.
100 µl Models: Remove and discard the white plastic flange using scissors or pliers. In doing so, be careful not to bend or crush the stainless steel tube. These pump models are designed to be as space efficient as possible for use in smaller animals, so they were designed without the translucent cap on the flow moderator. After catheter attachment, the catheter should cover about 4 mm of the length of the flow moderator (stainless steel tube).(see method)
Step 6. Fill the brain infusion assembly with the solution to be delivered. To do this, attach a syringe containing this solution to the remaining catheter tubing and connect the tubing to the free end of the flow moderator (as in photo).
Step 7. Fill the osmotic pump with the solution to be delivered, using the recommended procedure described in the ‘Filling and Priming’ section. Be certain that the fill volume is greater than or equal to the volume specified on the instruction sheet for that model.
Step 8. Fit the filled osmotic pump to its flow moderator. The pump and brain infusion assembly should now be completely filled and free of air bubbles.
Step 9. To minimize the chance of clotting, prime the filled brain infusion assembly with attached osmotic pump in sterile saline. (See Priming for more information.)
Step 6. Photo of brain infusion assembly with extra catheter tubing for filling
Step 10. The pump and brain infusion assembly are now ready for implantation.
Step 10. An ALZET Osmotic Pump and components of the ALZET Brain Infusion Kit 2 assembled prior to implantation.
Note: the ALZET pump and flow moderator are not included with ALZET Brain Infusion Kits. They must be purchased separately.