Intracranial Infusions
SEE PDF AT BOTTOM FOR MOST RECENT VERSION OF THIS PROTOCOL
Intracranial infusions (hereafter just infusions) is the process of infusing pharmacological agents into specific brain regions through prior surgically implanted cannula and use of an infusion pump, to temporarily alter neuronal activity, and measure its effect on behavior. This protocol explains each of the parts needed to do infusions as well how to actually do it and general tips/warnings. Reference section for further reading is included at the end.
First understand that while the number of parts needed to do infusion are limited it is a delicate and difficult process, that requires a lot of training to get it right, so be patient.
Most importantly before beginning infusions that will be kept as data points, the rats must be very very well habituated to the infusion process. This means for at least 5 days before infusions rats should undergo habituation described below. If rats are not habituated it greatly increases the difficulty of performing the infusion.
Part List:
1-2 10ul Hamilton syringes with needles (Hamilton company)
guide cannula (Plasticsone)
internal cannula (Plasticsone)
dummy cannula (Plasticsone)
cannula tubing (Plasticsone)
70% ethanol
Syringe Pump (World precision instruments)
Sterilized mineral oil
Kim tech wipes
Gloves
lab coat
Cannula:
Cannula consist of small metal wires or tubes with pieces of attached plastic. There are three types of cannula internal, guide, and dummy. Guide cannula are the cannulas that are surgically implanted into the brain and consist of small metal tubes surrounded by plastic. Internal cannula are inserted into the guide cannula the infusion drug is pump out of the internal cannula. Dummy cannula are kept inserted into the guide cannula anytime there is not a infusion being performed to keep the guide cannula tool clear of blood or CSF. It is important to keep the dummy cannula in the guide cannula as much as possible otherwise the brain is exposed to the air which increases the chance of infection.
Pump:
The infusion pump is a delicate piece of equipment(YOU MUST BE CAREFUL NEVER TO DROP/SLAM THE INFUSION PUMP). The pump uses a computer controlled back-plate to slowly push the plungers into the syringes to deliver the exact amount of infusion liquid out the tips of the internal cannula. The pump has many settings, to set to deliver the exact amount and rate of drug out the internal cannula. The relevant ones for the retrieval practice inactivation study will be explained throughout the protocol (ALWAYS CHECK THE PUMP TO MAKE SURE ITS SETTINGS ARE THE ONES REQUIRED FOR YOUR EXPERIMENT)
Main Window
This is the infusion pump main window check to make sure the syringe/rates/target setting are correct.
The arrow keys move the backplate forward or backwards, Be sure when moving the backplate forward not to bend the plungers or try to push them past the 0 ul mark on the syringe.
The run key takes you to the infusion window and starts an infusion.
Infusion Window
This is the infusion window on the infusion pump it gives you the details of the infusion.
The syringe figures on the left and text tells you how long the infusion has been going and how long is left as well as if it is actually infusing.
The blue window give you the general information about the infusion.
The arrow keys here also move the backplate.
The run button starts the infusion as soon as you press it.
Syringe/Needles: In addition to the pump syringes with needles are attached, they are locked into the infusion pump and it is the pushing of the backplate on the syringe plungers that push mineral oil through the syringe and needles down the tubes to push the infusion liquid out of the internal cannula and into the brain.
Preparing mineral oil:
Mineral oil is placed in the syringes, and tubing instead of air, as air is compressible, meaning that when the plungers start to push the air the air molecules can take time to press into the next set of molecules and so and so forth, this means it takes longer for the air to push against the infusion liquid and have it exit the internal cannula. Mineral oil is in comparison much less compressible so that molecules immediately push against the next set speeding the infusion process.
1. Take the mineral oil and pour out a fair amount into a large weigh boat
2. Next take a 1 ml syringe and place a syringe filter on the end then a 19g needle on the end.
3. Take on of the large helping hand stands and place the end of the sryinge in the mineral oil and pull the plunger out
4. Place the plunger in one of the arms of the helping hand stands.
5. It will take quite a bit of time for the mineral oil to fill the needle due to its high viscosity
6. After the needle is loaded remove the syringe filter and 19g needle and place a 23g needle on the end and cap it.
7. Place any unused mineral oil back into the mineral oil bottle.
Filling the tubes/syringe with mineral oil.
1. To load the tubes and syringes with mineral oil. Remove internal cannula from the end of the tubing if they are attached.
2. Place the needle with the sterilized mineral oil into one of the tubes, be sure to place the needle a few mms into the tubing other wise the injection pressure will pus the tubing off the end of the needle.
3. Next gently pull the plunger from the syringe that is connected to the tubing with the needle on it out.
4. Gently push on the mineral oil needle plunger and fill the tubing and syring completely with mineral oil, there should no bubbles of air.
5. Next while still pushing in mineral oil put the mineral oil needle out of the end of the tube.
6. Place the internal cannula back on the end of the tubing make sure the end of the tubing is flush with the internal cannula
7. Next take a small amount of mineral oil and place it on the end of the Hamilton syringe plunger and place the plunger back into the syringe
8. Place the plunger all the way down to the zero mark on the syringe.
9. Some mineral oil should flow out of the internal cannula just wipe it way with a kimtech wipe.
Habituation: Every other day post surgery the rats dummy cannula should be removed, placed in 70% ethanol, then taken out allowed to air dry and placed back in the rats guide cannula.
Preparing for infusion:
1. Check the tubing for any air bubbles, a few small ones are tolerable but any more than 5+ or large ones, then the mineral oil in the tubing should be replaced (described above).
2. check what infusion is needed for the rat and acquire the desired infusion liquid. Always ask if you are unsure.
3. Push the plungers on the needles all the way to zero and wipe away any liquid/mineral oil that comes out.
4. Next place the internal cannula into 70% ethanol for at least 20 seconds to sterilize them.
5. place the internal cannula into the infusion liquid and then gently pull back the plungers to 3 or 4 ul on the 10ul syringe.
6. This loading of the liquid takes awhile so wait to see no more liquid/mineral oil movement before moving the internal cannula out of the infusion liquid.
7. If you pull the plungers out while the cannula are not in the infusion liquid you will pull air into the cannula affecting the quality of the infusion.
8. After loading the infusion liquid return it to its proper storage spot.
9. Next using the arrow keys on the infusion pump position the back plate right up to the end of the plungers.
10. Next run the infusion until you see small amount of liquid come out of the internal cannula.
11. Stop the infusion and be sure to hit the restart button.
12. Gently wipe away the small amount of liquid off the internal cannula.
13. Position the infusion pump near the edge of the table so that you have a enough room for the tubes to reach the rat in your lap.
Infusion procedure:
1. Place habituated rat onto your lab and carefully remove the dummy cannula and place the dummy cannula into 70% ethanol
2. Take the prepped internal cannula attached to the pump via tubing and gently place it into the guide cannula, making sure that it is completely flush and periodically checking this throughout the infusion process.
3. Hit run on the infusion pump and let the pump fully pump the entire amount of the agent or PBS into the brain.
4. After the infusion has stopped a 1 minute post infusion time begins this time is critical to let the infusion liquid diffuse away from the tips of the internal cannula into the tissue.
5. After the 1 minute diffusion time is up, slowly draw out the internal cannula so as to minimize the vacuum force, which can pull the infusion agent out of the brain.
6. Take the dummy cannula out of the 70% ethanol and make sure there is no ethanol sitting on the cannula before placing them back into the guide cannula.
7. After this rerun the infusion program and check to see liquid comes out of the tips, this a check to make sure the infusion actually worked. If no liquid flows it is likely the needle became blocked and no liquid was actually injected into the brain. The infusion will need to be repeated.
8. Always be sure to push out any remaining liquid out of the tubes after the infusion is completed.
Troubleshooting:
Retrieval Practice specifics:
4% W/V Lidocaine is kept in the freezer each tube as number on top that matches the name of rat only use that tube for that rat.
The PBS is kept in the fridge in tubes with numbers on top.
The infusion volume is 0.5 ul at a rate of 0.5ul per minute so the infusion will take one minute.