Sean Lauber:Mouse Endotracheal Intubation
Arrange with Jane Ann before you plan to intubate. She has a calendar and you need to mark down when you plan on using her equipment. Remember that it is HER equipment and you must show respect for it. When you're done using the equipment, clean it with vitrol in the CAF, dry the canulas with the pressurized air, and put the stuff back where you found it. If you killed one of the two batteries for the ottoscope, make sure you arrange to have it charged (unscrew the ottoscope and plug it in overnight). Once the battery is charged, write the date that it was charged on a piece of tape and place it in the spare battery box. Please remmeber to charge the battery. It is horrible if you're downstairs in the CAF and you've run out of battery power, because at this point you cannot continue your experiment.
1. Collect the necessary components for the intubation:
The rack The ottoscope The ottoscope fittings The canulas The spare battery Masking tape PBS Virus Calculations for virus (if you're making the virus in the CAF) Ependorffs 2x P200 pipettes Filter tips Regular tips Forceps Isoflurane Timer (optional for timing how long it takes for the mice to go down)
2. Prepare your virus
3. Prepare the rack and accessories (place folded tape over the rack, set the rack at the furthest notch, assemble the ottoscope, remove the canula from its box)
4. Prepare your tester pipette for confirming entry in trachea (load a p200 with a non-filter tip and set it to 150 ul, draw up about 75 ul of PBS and draw air the rest of the way)
5. Load 50 ul into the other pipette with a filtered tip, this will be your virus pipette
6. Place 1 mouse in the isoflurane chamber (at the 5th setting)
7. Once the mouse starts showing hunched breathing (about 2 minutes) wait another 30 seconds. Time this step if necessary.
8. Remove the mouse from the chamber by the base of the tail (for better stabilty in arranging the head) and place it on the rack, moving its head to hang it from its teeth using the thinner of the two strings. You want the chest pointing upwards and you want the mouse to be straight
9. Secure the tape on around the chest of the mouse
10. Turn the ottoscope on and pick up the forceps
11. Lift the mouse's tongue with the forceps and enter the mouse's mouth with the attached ottoscope piece (don't jab it in too far or you'll injure the mouse). You want to go fairly deep and cleanly - make sure the tongue is not in the way.
12. While looking through the magnifier on the ottoscope, and with a lifting (of the tongue) and pulling motion, draw back the ottoscope (off the tongue) until you see the opening to the trachea (the vocal cords should open and close as the mouse breathes). This is a difficult step to get right so really try to perfect this.
13. Once the vocal cords are located, grab the canula and insert it into the trachea (easier said then done) - again, do not put it too deep down the trachea. If you cannot see the vocal cords it's because you didn't go deep enough in the previous step or you pulled back too qucikly that you missed it. Try again.
14. Hold the canula with one hand as you turn off and place the ottoscope down (conserving battery life can be critical)
15. Remove the insert in the canula and get the tester pipette to confirm you've gained access. Make sure the pipette tip is forming a good seal so you can note the breathing by the movement of the PBS. If the mouse stops breathing you can press on its chest (sometimes works) or remove the pipette tip and wait for it to start breathing again, and then put the tester pipette tip back to check for entry. Occasionally the PBS in the tip will splash around and will not move up and down with the mouse's breathing. Simply repace the tip and get new PBS.
16. Once you've confirmed you're in the trachea you can trade for the virus pipette and eject the liquid into the canula. The mouse should inhale it almost instantly. Watch the opening in the canula to make sure it's all gone before removing it. The mouse should inhale it deeply and there shouldn't be any trouble. You should never force the liquid into the trachea by ejecting the virus directly into the trachea. You want to slowly eject the liquid toward the base of the canula opening so the mouse sucks it up. The mouse should be in a state of anasthesia that it breathes deeply and inhales the liquid in one or two breaths. Make sure you get this right!
17. Move the mouse to a recovery cage until you can move it back to its home. Warm the mouse if you see it's struggling.
18. Clean up everything with vitrol, especially the canula (then rinse with water). Once upstairs dry the canula with the pressurized air and place everything back
19. If the battery needs charging, do it now. If it's Friday, leave yourself a note to remind you to do it Monday.
When you get really good you should be able to do about 10 mice in 30 minutes. I'm talking really good.
Some pointers. If you mess a mouse up (it will happen), it's okay to put the mouse back in the chamber for 30-60 seconds and then try again. The worst I've done is repeat a mouse about 5 times. I didn't expect him to take me out for drinks after (his trachea did not look good) but if this is what it takes then so be it. But be warned that repeating a mouse means the trachea will appear bloody and/or mucousy - reducing visibility of the vocal cords. Learn how to get it right the first time! Once you get good you can do multiple mice in a chamber and be able to pump them out every 1-2 minutes or so. It can go very quickly if you have the timing right, but can be a huge disaster if you start messing them up. If you have multiple mice in a chamber and you have to keep repeating one, start alternating. You don't want one mouse to be in the chamber for too long or they will be so deep that they won't inhale the virus effectively. This is why you need to start off slow, develop your skills, and then try to be more efficient. You must learn how to do each mouse in less than 30 seconds. It sounds crazy but this is the only way to properly do this. The real trick is to ensure that you're in the trachea and you see nice movement of the PBS in the tester pipette. Do not, nay, NEVER proceed if you don't see this. You're wasting your time and likely about to waste a mouse. If you do this, and the mouse inhales part of the virus, try best to reclaim what's left (flush it out with PBS) and try to do it again with 50 ul (top it off with PBS). The mouse can handle a bit more than 50 ul of liquid in the lungs, but I wouldn't try more than 100 ul total. I have had to do this once and I regretted it. It compromises the experiment and the safety of the mouse. They're small and have about 1 ml lung capacity, so imagine inahling about 10% of your lung capacity in liquid (about 1 L of water). Not a good thing.
And now for words of encouragement. This is not easy. In fact, it is probably the most difficult technical thing I have ever done. I didn't learn this overnight, it took me about a year and a hundred mice before I could get consistent results. In the beginning I thought I wouldn't get it. Jane Ann and I spent about an hour before I was able to do one. When I went back later on my own I couldn't get it. I thought Jane Ann would have to do it for me for the rest of my project. What you need to do is practice on a weekly basis until you've got it. In my case it took about 10 to 20 sessions. Results will vary but stick to it. You're about to become one of the few people IN THE WORLD to be able to intubate a mouse. Definitely something to be proud of, so get to being proud!