The overall goal of this procedure is to implant mini-osmotic pumps in order to deliver therapeutic agents to the brain and then to deliver tract tracers to study modifications on corticospinal tract conductivity. This method can help answer key questions in the field of neurorestorative research such as how long distance connections within the central nervous systems are altered in the presence of potential therapeutic agents. The main advantage of these techniques is that continuous delivery of the therapeutic agent grants the reproducibility of results whereas the tract tracers are very reliable agents to follow morphological changes in long distance connections.
Generally, individuals new to these methods will struggle because precision and surgical abilities are essential in order to prevent brain damage as a consequence of the various interventions. I will be demonstrating the procedure along with Jeismar Carballo, a graduate student from professor Hammond's laboratory. Begin by preparing the mini-osmotic pump in a sterile environment.
First, use forceps to hold the brain infusion cannula upside down. Then, add two drops of cyanoacrylate adhesive and introduce one spacer disk for drug delivery to the ventricle. Next, cut the catheter to 2.5 centimeters and carefully connect it to the flow moderator.
Then, to prevent the formation of air bubbles, use a 2 milliliter syringe connected to a needle to fill the pump until a small amount of solution escapes the pump. Now, carefully fill the flow moderator and catheter, ensuring that there are no bubbles inside the catheter. Then, fit the flow moderator inside the pump.
Once filled, carefully connect the cannula to the end of the catheter then place the pump in a container of sterile saline solution and leave it at 37 degrees Celsius overnight. After anesthetizing the animal according to approved protocols and confirming anesthesia by lack of a response to a hind-paw pinch, place the animal in a stereotaxic frame. Cover the eyes with protective ointment to prevent drying.
Also, cut the fur over the head as close to the skin as possible and clean the skin with 70 percent ethanol and a disinfectant with antibacterial and fungicide properties. After using a scalpel to open a 1 centimeter incision slightly to the right of the midline, expose the skull and clean it with a cotton-tipped swab soaked in 70 percent ethanol. After sundering the stereotaxic arm over bregma, move the stereotaxic arm to the coordinates to target the ventricle.
Mark this point on the skull. Gently drill the skull at this point with the drill angled at 45 degrees to prevent accidental damage to the brain. Repeatedly drill for a few seconds and then check the depth of the hole.
Stop drilling once the skull has been thinned but still not fully penetrated then break the meninges with the tip of a sterile needle until access to the brain is clear. Next, introduce straight forceps under the skin of the animal in an anterocaudal direction and use the forceps to open the space under the skin in the back of the animal where the pump will be implanted. Introduce the pump into the back of the animal leaving the catheter and cannula outside.
Carefully place four small drops of glue on the cannula next to the needle. Now, carefully introduce the needle through the skull, without moving it sideways. Hold it in position for 15 to 30 seconds until the cannula is completely attached.
It is important to be gentle and accurate when introducing the pump needle and to make sure the cannula is properly fixed to the skull to ensure it completes the delivery period without being removed by the animal. Then, place one finger over the removable tab and use scissors to cut it off at the narrow part. Finally, close the incision with five zero suture and disinfect the closed incision with povidone-iodine solution.
Move the animal into a clean cage and monitor it until it has regained sternal recumbency. The animal should be singly housed during drug administration. After preparing, anesthetizing and placing the animal in the stereotaxic frame as before, open the skin by cutting through the incision used for pump implantation then hold the cannula with a surgical clamp and pull it out as shown.
It should be easily removed from the skull. Stop any bleeding by holding a cotton-tipped swab to the site for one to two minutes. Grasp the catheter and remove the pump by pulling the catheter and pushing the pump at the same time.
After removal of the pump, close the wound as before, and apply a few drops of povidone-iodine. Transfer the animal to a clean cage and monitor it until mobile. Ten days after removal of the mini-osmotic pump, anesthetize the animal, return it to the stereotaxic frame, and open the head wound.
Clean the skull with 70 percent ethanol. Properly fit a five microliter glass syringe on the vertical holder of the stereotaxic device. Orient the syringe directly over bregma and move to the desired coordinates to inject the motor cortex.
Make a mark at this point with a marker with a thin tip. Next, move the syringe to the second point and make a small mark at this point. Carefully drill the skull at both points while holding the drill at a 45 degree angle.
Again, do not fully perforate the skull and use the tip of a needle to remove the last of the bone. Load the syringe with 600 nanoliters of the tracer. Then adjust the position of the needle to a 45 degree angle on the right side of the skull.
While looking through a surgical microscope, position the tip of the needle right in front of the hole then insert the needle into the brain to a depth of 1.5 millimeters. The speed shown is just for the purposes of demonstration. Normally, wait 30 seconds between each 0.5 millimeter step.
Allow the needle to remain in place for 30 seconds to one minute. As you have seen, the penetration of the needle during tract tracer injection is from the mantle to avoid targeting incorrect areas which might lead to confusing results between individuals. Slow removal of the needle will prevent backflow of the tracer.
Then, inject 300 nanoliters of tracer in three 100 nanoliter steps with each step separated by a 30 second interval. After the last injection, allow the syringe to remain in place for 30 seconds to one minute before withdrawing the syringe slowly. Position the tip of the syringe over the second hole.
Insert the needle as before and then repeat the injection with the remaining 300 nanoliters in the syringe. After the second injection, withdraw the syringe and close the wound with five zero suture. Again, place the animal in a clean cage and monitor the animal while it recovers from anesthetic before returning it to the housing room.
Animals were subjected to 30 minutes of middle cerebral artery occlusion to induce a lesion in the left striatum. Three days later, recombinant human erythropoietin was administered via mini-osmotic pumps for a period of 30 days. Cascade blue was injected into the infarcted hemisphere and biotinylated dextran amine was injected into the contralateral hemisphere.
Fibers were followed to the facial nucleus. Biotin dextran amine injected in the contralesional motor cortex is detected in corticobulbar fibers. Fibers to the ipsilesional and contralesional hemisphere were counted and expressed as a percentage of total labeled fibers in the corticospinal tract.
Erythropoietin increased fiber crossings in direction to the contralesional facial nucleus. Motor behavior showed an improvement of hand grip strength compared with pre-ischaemic baseline after recombinant human erythropoietin infusion. Motor coordination, as assessed by the rotarod test, was also improved after recombinant human erythropoietin infusion.
Once mastered, this technique can be done in less than 30 minutes per surgical procedure if it is performed properly. While attempting this procedure, its important to remember to maintain adequate sterility during pump preparation and during surgery to prevent brain infections. The use of anterograde and retrograde tract tracers is a safe non-expensive approach that allows to understand the connectivity of the brain in both health and disease.
After watching this video, you should have a good understanding of how to implant and remove mini-osmotic pumps and or perform tract tracer injections.
