Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

This protocol can be used to perform convection-enhanced delivery in small rodents using a step catheter system for the adjustable uniform perfusion of target brain regions. The advantage of convection-enhanced delivery is that it allows delivery of substances into the region of interest bypassing the blood-brain barrier with little tissue damage or reflux. This technique is primarily useful for delivery of therapeutic substances such as antibodies into the brain.

And can therefore be used to target many neurological conditions. Demonstrating the procedure will be Michal Beffinger, a post-doc from my laboratory. Begin by cutting a piece of fused silica capillary tubing with an inner diameter of 0.1 millimeter and a wall thickness of 0.325 millimeter to a length of 30 millimeters.

After examining the tubing for cracks use a microforge to heat polish the ends to ensure the tubing openings have a smooth surface. Next, mount a 27-gauge needle onto a 10 micro liter syringe and place the syringe in a stereotactic robot. Use the robot to move the syringe over a hard surface and touch the surface with the needle tip.

After noting this position elevate the needle to enable placement of the fused silica capillary inside of the needle such that 20 millimeters of the capillary protrudes from the needle. Use a pipette to evenly spread two microliters of high viscosity cyanoacrylate adhesive over the capillary starting from the metal needle and finishing ten millimeters above the lower end of the capillary. Use the stereotactic robot to lower the metal needle until the tip of the needle is one millimeter over the reference surface to fix the fused silica capillary in the metal needle forming a one millimeter step from the tip of the metal needle.

Remove any excess glue forming at the end of the metal needle to avoid planting the catheter step. Check the tip under a microscope to confirm that all of the excess glue has been removed. Then wait 15 minutes for the glue to harden and remove the syringe with the catheter from the robot.

For step catheter testing cut solidified 0.6%agarose gel into 20 by 20 millimeter blocks and manually fill the step catheter syringe with 10 microliters of filtered 0.4%trypan blue solution. Using the stereotactic robot, dispense one microliter of dye at 0.2 micro liters per minute to assess the sealing of the step of the catheter during the fixation procedure. The trypan blue solution should be visible solely on the tip of the catheter.

Wipe away the dye with a paper tissue and place an agarose block in the stereotactic robot. Calibrate the robot said that the tip of the catheter is referenced against the surface of the agarose block. Set a proportion sequence of injection volumes according to the specific experimental plan.

To inject the solution into the murine caudate putamen, set the injection to a one millimeter frontal and one and a half to two millimeter lateral from bregma position at a depth of 3.5 millimeters. When the needle is in position start the convection enhanced delivery procedure and inject five microliters of trypan blue solution into the agarose block. Assess the shape of the trypan blue cloud in the agarose and for potential leakage along the catheter tract.

No major backflow over the tip of the metal needle should be visible. After the injection leave the catheter in place for two minutes before retracting the needle at one millimetre per minute to ensure a proper dispersion of the fluid into the brain and sealing of the injection tract during the removal. Place a new agarose block into the robot and start a second injection of one microliter at 0.2 micro liters per minute to assess clogging of the catheter within the agarose.

The trypan blue should again form a cloud from the tip of the catheter immediately after the start of the injection. Then assess whether the leftover volume in the syringe corresponds to 3 microliters as any variations might indicate a leakage of fluid through the catheter mounting or syringe plunger. For antibody injection into the murine striatum confirm a lack of response to skin pinch in an anesthetized Mouse and shave the head with a hair trimmer.

Disinfect the skin with cotton swabs soaked in iodine solution. Use a scalpel to make a 10 millimeter skin incision along the cranial midline finishing at the eye level. Fix the mouse in the stereotactic frame using the nose clamp and ear bars, taking care that the skull surface is horizontal and tightly secured.

Place the syringe in the stereotactic robot and synchronize the drill bit with the tip of the catheter on a reference point. Use forceps to retract the skin and localize bregma on the skull surface. Reference bregma in the software using the tip of the drill bit and move the drill to a one millimeter frontal and two millimeter lateral from bregma position.

Drill a burr hole taking care not to damage the dura mater and move the syringe over the burr hole. Dispense 0.5 to one microliter from the syringe to ensure that no air bubbles are left in the catheter. Start the convection enhanced delivery as demonstrated, observing the skull surface for any traces of fluid backflow from the injection spot.

At the end of the delivery and catheter removal start the injection pump at 0.2 micro liters per minute to check for evidence of catheter clogging during the injection. If no clogging occurred a droplet of injection mix should immediately be observed coming from the catheter tip. In this image of a cloud of trypan blue forming after the injection of one microliter of dye at 0.5 micro liters per minute using a convection enhanced catheter as demonstrated, no reflux along the needle tract was visible over the beginning of the catheter step.

And the dispersed cloud formed a desired spherical shape. In this image using a blunt end needle however, significant reflux could be observed. Notably, convection enhanced delivery enables the perfusion of large volumes into the murine brain in a uniform and less tissue damaging manner compared to conventional injection.

In both types of delivery there is a typical distribution profile of antibody and dextran particles over the corpus callosum. However, the dispersion profile of the injected antibody is more diffused than is observed for the high molecular weight dextran after convection enhanced delivery, exemplifying differences in distribution between different infusates. As the catheter can potentially clog during the brain infusion, it is important to check it immediately after by slowly dispensing a small volume of infusate.

This procedure can serve as a way to deliver pharmacologically active compounds to the brain and should be followed by close monitoring of disease symptoms and expected side effects. This technique enables the infusion of a precise brain region with therapeutics including antibodies, opening new possibilities for the development of central nervous system targeted therapies.