Convection-Enhanced Delivery of Antibodies into a Mouse Brain

Overview

This video demonstrates a convection-enhanced delivery (CED) technique for antibody delivery into the mouse brain. The skull of an anesthetized mouse is exposed, and a burr hole is created at the target area for antibody delivery. A step catheter containing antibodies is inserted at the target site, and via an infusion pump, a pressure gradient is created to deliver antibodies at a controlled rate.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of the Step Catheters

1. Preparation of a fused silica tubing for the step of the catheter
   1. Cut the fused silica capillary with an inner diameter of 0.1 mm and wall thickness of 0.0325 mm tubing to a length of 30 mm.
   2. Examine the tubing for cracks and heat polish the ends using a microforge to ensure the tubing openings have a smooth surface.
2. Fixation of the inner tube in a metal needle
   1. Mount a 27 G needle on a 10 μL syringe and place the syringe in a stereotactic robot.
   2. Using the robot, move the syringe over a hard surface and touch it with the needle tip. This position should be noted or saved in the software because it will serve as a reference surface for setting the length of the catheter step.
   3. Elevate the needle to enable the fused silica capillary to be placed inside of the needle.
   4. Place the fused silica capillary in the needle such that 20 mm of the capillary is protruding from the needle.
   5. Using a pipette, evenly spread 2 μL of high-viscosity cyanoacrylate adhesive over the capillary, starting from the metal needle and finishing 10 mm above the lower end of the capillary, as depicted in Figure 1.
   6. Using the stereotactic robot, lower the needle until the tip of the metal needle is 1 mm over the reference surface. This way, the fused silica capillary will be fixed in the metal needle, forming a 1 mm step from the tip. Remove any excess glue forming at the end of the metal needle to avoid blunting the step.
   7. Wait 15 min for the glue to harden, and remove the syringe with the catheter from the stereotactic robot. Confirm that all excess glue has been removed at the step by checking the tip of the catheter under a microscope.
3. Testing the step catheter using a block of agarose
   1. Prepare 0.6% agarose solution in phosphate-buffered saline (PBS) in a conventional gel tray and wait until it polymerizes. Cut the agarose in approximately 20 mm x 20 mm blocks. Until use, keep the blocks immersed in PBS.
   2. Manually fill the step catheter syringe with 10 μL of 0.4% solution of filtered trypan blue.
   3. Using the stereotactic robot, dispense 1 μL at 0.2 μL/min to assess the sealing of the catheter step during the fixation procedure. Trypan blue solution should be visible solely on the tip of the catheter. Wipe it off with a paper tissue.
   4. Place the agarose block in the stereotactic robot and calibrate the robot so the tip of the catheter is referenced against the surface of the agarose block.
   5. Program the injection parameters for CED.
      1. For the injection volume of 5 µL, use the following steps: 1 µL at 0.2 µL/min, then 2 µL at 0.5 µL/min and 2 µL at 0.8 µL/min. Adjust the final injection volume according to the specific experimental plan by proportionally changing the duration of each of the steps.
      2. To inject the solution into murine caudate putamen (striatum), perform such injection in a position 1 mm frontal and 1.5–2 mm lateral from bregma at a depth of 3.5 mm.
      3. After the injection, leave the catheter in place for 2 min and then retract at 1 mm/min to ensure proper fluid dispersion in the brain and seal the injection tract during catheter removal.
         NOTE: All parameters can be programmed into a single script depending on the specific stereotactic robot.
   6. Start the CED procedure and inject 5 μL of trypan blue solution into the agarose block.
   7. Assess the shape of a cloud of trypan blue in the agarose and potential leakage along the catheter tract. Trypan blue should form an ellipsoid or a round cloud with the center around the catheter tip and a diameter of at least 1 mm. No major backflow over the tip of the metal needle should be visible.
   8. Place a new agarose block and start a second injection of 1 µL at 0.2 µL/min to assess the catheter's clogging with the agarose. Trypan blue should again start forming a cloud from the tip of the catheter immediately after the start of the injection.
   9. Assess whether the leftover volume in the syringe corresponds to 3 µL. Any variations might indicate fluid leakage through the catheter mounting or syringe plunger.
   10. If all the test injections are successful, the catheter is well sealed and straight, and no trypan blue solution is observed from other spots than the catheter tip, wash the catheter with deionized H₂O (dH₂O) until no traces of trypan blue are visible and then wash ten times as follows: 70% ethanol and 100% ethanol followed by flushing again with 70% ethanol and clean deionized water.
   11. Store the catheter under dry conditions.

2. Convection-enhanced Delivery of Antibody Solution into the Murine Brain

NOTE: Depending on local animal welfare regulations, various types of anesthetics, analgesics, and antibiotics can be implemented for this procedure. This protocol describes the use of injection anesthesia. Inhalation anesthetics such as isoflurane can also be used by mounting a nose mask on the stereotactic frame. In addition, we recommend adding antibiotics to the drinking water for infection prophylaxis.

1. Surgical setup
   1. Prepare anesthetics and antidote solutions. Mice can be safely anesthetized using a three-component anesthesia containing fentanyl (0.05 mg/kg), midazolam (5 mg/kg), and medetomidine (0.5 mg/kg) diluted in sterile distilled water (dH₂O). We perform a two-step wake-up procedure using two antidote solutions, one containing flumazenil (0.5 mg/kg) and buprenorphine (0.1 mg/kg) in sterile dH₂O (first antidote solution). The second one has atipamezole (2.5 mg/kg) in sterile dH₂O (second antidote solution).
   2. Prepare an analgesia solution containing carprofen (5.667 mg/kg) diluted with sterile dH₂O.
   3. Clean the stereotactic frame, heating pad, and elements of the stereotactic robot. Bear in mind that not all the robot parts can be cleaned without risk of damage. Refer to the robot manual for details on cleaning and preparing for usage.
   4. Assemble the syringe with the step catheter and flush it multiple times with dH₂O, 70% ethanol, and 100% ethanol, followed by flushing again with 70% ethanol and dH₂O. Finally, flush the syringe with PBS or other buffers to prepare the solution for intracranial injection, e.g., artificial cerebrospinal fluid. The syringe plunger should move smoothly and freely during the procedure.
   5. Calibrate the stereotactic robot software with the stereotactic frame.
   6. Test the stereotactic robot software by ensuring that the robot arms move freely, that the injection pump is appropriately connected, and that it can perform the CED procedure without any disturbances. This includes testing robot movement, ramping injection, checking the 2-minute waiting step, and checking the speed of catheter retraction. All the parameters should fit the preprogrammed CED procedure described in point 1.3.5.
   7. Insert the drill bit in the drill. Sterilizing the drill bits before use is recommended.
   8. Prepare antibody solution using PBS or other buffer solutions such as artificial cerebrospinal fluid (aCSF). 1 to 20 µg of antibody in 5 µL can be injected in a single CED procedure. Other volumes and protein amounts should be tested before performing the experiment. Be aware that using high-viscosity solutions might lead to catheter clogging.
   9. Manually load the syringe with the diluted antibody.
2. Antibody injection by CED into the striatum
   1. Weigh the mouse and inject the three-component anesthesia solution into the peritoneum according to the body weight. Note the injection time. Transfer the mouse to a separate cage heated with a heating pad.
   2. Observe the mouse to determine when the sedation starts. When the mouse stops moving, apply ophthalmic ointment on the eyes to protect the cornea from drying out during the surgery. Full sedation usually starts 10–15 min after the injection of the three-component anesthesia solution.
   3. Check pain reactions using the pinch-reflex test to ensure the animal's complete anesthesia.
   4. Shave the head using a hair trimmer.
   5. Disinfect the skin with cotton swabs soaked in iodine solution. Scrub the skin three times in a circular motion.
   6. Make a 10 mm skin incision along the cranial midline, finishing on the eye level using a scalpel.
   7. Fix the mouse in the stereotactic frame using the nose clamp and ear bars. Ensure that the skull surface is horizontal and tightly secured. Besides correct anatomical navigation, it is also crucial to avoid tilting the skull during the drilling and the CED procedure.
   8. Place the syringe in the stereotactic robot.
   9. Synchronize the drill bit with the tip of the catheter on a reference point. The relation between the position of the drill and the syringe must be precisely determined in the software so the injection can be performed in the desired anatomical region of the brain.
   10. Retract the skin using forceps and localize the bregma on the skull's surface.
   11. Reference Bregma in the software using the drill bit tip.
   12. Move the drill to a position 1 mm frontal and 2 mm lateral from bregma and drill a burr hole. Be careful not to damage the dura mater.
   13. Move the syringe over the burr hole.
   14. Dispense 0.5–1 μL from the syringe to ensure no air bubbles are left in the catheter.
   15. Start the CED program described in point 1.3.5. Observe the skull surface for any traces of fluid backflow from the injection spot. Monitor the breathing rate of the animal.
   16. Once the CED program is over and the catheter is withdrawn from the brain, start the injection pump at 0.2 µL/min to check for catheter clogging during the CED. If no clogging occurred, you should immediately see a droplet of injection mix from the catheter tip.
   17. Before reusing or storing the catheter, visually examine the catheter step under a microscope for any signs of damage or wear and clean it as in step 1.3.10.

Representative Results

Figure 1: A schematic drawing depicting the application area of the adhesive. The upper 10 mm of the fused silica tubing is inserted into the metal needle. Apply the glue on the 10 mm tubing starting from the tip of the metal needle.

Materials

Name	Company	Catalog Number	Comments
10 μL syringe	Hamilton	7635-01
27 G blunt end needle	Hamilton	7762-01
Agarose	Promega	V3121
Atipamezol	Janssen
Bone wax	Braun	1029754
Buprenorphine	Indivior Schweiz AG
Carprofen	Pfizer AG
Dental drill bits, steel, size ISO 009	Hager & Meisinger	1RF009
Ethanol 100%	Reuss-Chemie AG	179-VL03K-/1
Fentanyl	Helvepharm AG
FITC-Dextran, 2000 kDa	Sigma Aldrich	FD2000S
Flumazenil	Labatec Pharma AG
Formaldehyde	Sigma Aldrich	F8775-500ML
High viscosity cyanoacrylate glue	Migros
Iodine solution	Mundipharma
Medetomidin	Orion Pharma AG
Microforge	Narishige	MF-900
Midazolam	Roche Pharma AG
Ophthalmic ointment	Bausch + Lomb	Vitamin A Blache
PBS	ThermoFischer Scientific	10010023
Polyclonal goat anti-rat IgG (H+L) antibody coupled with Alexa Fluor 647	Jackson Immuno
Scalpels	Braun	BB518
Silica tubing internal diameter 0.1 mm, wall thickness of 0.0325 mm	Postnova	Z-FSS-100165
Stereotactic frame for mice	Stoelting	51615
Stereotactic robot	Neurostar	Drill and Injection Robot
Sucrose	Sigma Aldrich	S0389-500G
Topical tissue adhesive	Zoetis	GLUture
Trypan blue	ThermoFischer Scientific	15250061
Water	Bichsel	1000004