Hello, my name is Miles Cunningham. Together with Ryan O'Connor and Sydney Wong, we will demonstrate the construction of a micro infusion system for continuous delivery of neuroactive agents. This work was done in the laboratory for neuro reconstruction at McLean Hospital, Harvard Medical School.
We would like to thank the Sydney rbe Foundation and Narsad for their generous support. We have found that Neuroactive agents can be reliably delivered to discrete brain sites through a pulled Bo silicate glass micro cannula with a tip diameter five to 10 fold smaller than that of conventional delivery cannulas. In this slide, we show a commonly used stainless steel infusion cannula next to a micro cannula we've fabricated here in this laboratory.
This reduction in size of the delivery cannula translates into a dramatic reduction in trauma to the brain region being studied. Many conventional methods in effect, produce a lesion of or within the area of interest, rendering the validity of any result questionable. You can see here in panel A the amount of disruption to the local area caused by a 0.32 millimeter cannula when compared to a 50 micron cannula in panel B.These low power photos are of wet mounts of unstained Vibram sections.
These same sections were then stained with h and e and imaged at higher power. To further illustrate the reduction in tissue damage with the micro cannula, the components of the microinfusion system are an zet mini Osmo pump with its stainless steel flow modulator in which the flange has been removed. Two to three centimeters of polyethylene tubing, a custom made BOA silicate micro cannula, and a 28 gauge guide wire, and this is the assembled microinfusion system ready for implantation.
A standard vertical pipette polar is used here to create micro cannulas with the desired specifications. Horizontal polars are also suitable. The Bora silicate tubing is positioned and secured by varying the temperature of the heating element and the tension of the pole.
You can reliably reproduce micro cannulas with specific diameters and long tapering shank links. The Bora silica tubing is heated and gentle pressure is applied to reduce the desired amount of bend to the tubing. This bend is made at a distance from the tip of the micro cannula that is appropriate for the depth of the brain target of interest and allowing for clearance above the skull.
The final length of this micro cannula distal tube, the bend is approximately nine to 10 millimeters. Apparently not satisfied with his result. Ryan reinserts the micro cannula to make a fine adjustment perfecting the bend angle there.
That's much better using a diamond pencil. Sydney demonstrates a method for cutting the boric silicate tubing approximately five millimeters proximal to the bend, holding the tubing firmly on a hard surface with the distal portion of the micro cannula free to hang over the edge. She scores the tubing circumferentially.
Then by applying gentle pressure, she snaps the tubing cleanly at the scored interface. The fine transparent tip of the cannula can then be colored with permanent ink to aid in visualization. During surgery under magnification, the tip of the micro cannula is cut with microdissection scissors to the desired diameter.
The tip diameter can be confirmed with an eye piece or a microscope. Radical alignment wires are prepared from 28 gauge stainless steel wire that is cut in approximately three centimeter lengths. Forceps are then used to create a small hook at the end of each wire about two millimeters in diameter.
The alignment wires are placed in a Petri dish along with two to three centimeter lengths of polyethylene tubing in the micro cannulas. The microinfusion system can be constructed with instruments common to most laboratories. Micro forceps, conventional forceps, surgical scissors, and hemostats are placed in a surgical drape for sterilization.
The instruments, the microinfusion system components, as well as several sheets of aluminum foil are sterilized using a standard autoclave. The flow modulator is carefully removed from its packaging and placed into a sterile Petri dish. It is held securely with hemostats and the plastic flange is removed from the stainless steel modulator tube with scissors.
The mini osmotic pump is also removed from its packaging and placed in the sterile Petri dish. The stainless steel tubing is inserted into the polyethylene tubing about two to three millimeters, and the two are sealed together using light activated epoxy. We use Lummi bond from my neural lab for delicate work, we fill a one CC syringe with Lummi bond and use a 27 gauge needle as the applicator under bright light Lumon cures in just a few seconds.
The mini osmotic pump is filled by holding it in an upright position, carefully injecting it using the filling needle provided by the manufacturer. We recommend observing the fluid volume that is injected into the pump and or weighing the pump before and after filling to ensure the pump is completely filled and is not defective. Note that we do not recommend reusing pumps.
The full modulator with its attached polyethylene tubing is then inserted within the mini osmotic pump. Fluid within the pump will be forced into the polyethylene tubing as the foam modulator is inserted. The initial distance of polyethylene tubing filled with the fluid is noted and should be observed to increase after the pump has been activated with incubation and sterile saline at 39 degrees Celsius after 12 hours of incubation.
The activated mini osmotic pump is submerged in sterile saline, which provides an air free environment for the final steps in construction of the microinfusion system. A syringe fitted with small gauge plastic tubing and filled with the same solution contained by the pump is used to displace air from the unfilled portion of the polyethylene tubing. Note that air within the micro cannula should be displaced.
Similarly, the distal end of the polyethylene tubing may require some dilation with micro forceps and the proximal end of the micro cannula is then inserted. The entire system is now free of air or dead space and can be removed from the saline bath and bought a dry with sterile cause. Luma bond is then applied to secure the micro cannula to the polyethylene tubing To decrease the flow rate of the mini osmotic pump By approximately one half, sterile hot paraffin is poured into a small Petri dish and proximal one half of the mini osmotic pump is briefly submerged.
Excess paraffin is blotted and the microinfusion system is placed back into sterile saline and stored until surgery. Just prior to surgical implantation. The guide wire is attached to the bend in the micro cannula.
The longitudinal axis of the guide wire is aligned with the portion of the micro cannula that will enter the brain tissue and is fixed with lumon. In that position. The microinfusion system can then be positioned on a stereotaxic instrument manipulator.
The guide wire is secured with a manipulator arm clamp, and the pump is tethered from above with a loop of sterile suture. This will support its weight and allow the micro cannula to be perfectly aligned perpendicular to the surface of the skull by carefully bending the guide wire with forceps, the microinfusion system can then be implanted using standard stereotaxic procedure. The boel tubing is secured to the skull surface with adhesive.
We use den mat Jaron to sculpt a low profile pedestal. The excess guide wire extending above the preparation is then cut flush with the surface of the pedestal. Using a cutting wheel attached to a high speed drill, the mini osmotic pump can then be placed subcutaneously between the animal scape.
The present method offers a number of advantages over traditional techniques. It can be custom built to accommodate the species and age of the experimental animal. The silica tubing can be fashioned to conform to the surface of the animal skull.
Therefore, allowing secure low profile fixation and precluding the need for a large cranial pedestal, the surgical wound can easily be closed over the streamlined microinfusion pedestal, thus reducing the risk of infection as well as discomfort to the animal, and securing the system to the skull requires less surface area. Therefore, additional or subsequent procedures may be performed without interference from a larger pedestal. Most importantly, though, this method reduces invasiveness and trauma to the local environment of the infusion site, therefore minimizing the observer effect.
That is the changes that the active observing will make on the phenomenon being observed.
