Basic protocol



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CURRENT PROTOCOLS IN NEUROSCIENCE
  APPENDIX 4 ANIMAL TECHNIQUES
    APPENDIX 4A High Precision Stereotaxic Surgery in Mice
      BASIC PROTOCOL: SITE-SPECIFIC CENTRAL MICROINJECTION IN THE ANESTHETIZED MOUSE



BASIC PROTOCOL: SITE-SPECIFIC CENTRAL MICROINJECTION IN THE ANESTHETIZED MOUSE

For applications in which the mouse does not need to be awake and behaving (e.g., biochemical or molecular biological assays) or where the injected substance has a relatively long duration of action (e.g., antisense oligonucleotides or viral vectors), this protocol describes the delivery of substances to specific regions within the brains of mice. First, mice are anesthetized and mounted to the stereotaxic frame. The skull is exposed and measurements of the skull features are taken. Finally, a hole is drilled in the skull above the target structure, the injection is carried out, the animal is sutured and allowed to recover. The entire protocol takes ~30 min per animal and does not require the construction of cannulae, wire plugs, or injection needles, making this a less time consuming technique than cannulation. However, this protocol induces stress in the animals and introduces a fresh trauma to the brain simultaneous with the injection.



Materials

Ketamine/xylazine cocktail (see recipe)


Hair shaver or depilatory cream (optional)
Ophthalmic ointment or mineral oil
Betadine
Bone wax

Mouse stereotaxic frame (Cartesian Research) equipped with:


Sighting scope
Drill with #74 bit
Monoinjector with 24-G Hamilton syringe (model #8800 for injections of up to 5 ul)
Heating pad
Forceps
Scalpel
Surgical scissors
Agricola-style retractors (Fine Science Tools)
Cotton swabs
Sutures

Anesthetize mouse

1. Anesthetize the mouse with an intraperitoneal injection of an appropriate dose of ketamine/xylazine cocktail. After the animal has reached full anesthesia, remove the fur from the top of the skull with a shaver or depilatory cream or by plucking with fingertips or a pair of forceps.



A dose of ~20 ul/g body weight is generally appropriate, however, mice of different strains, ages, and adiposities will have differing sensitivities to the anesthetic. The total volume of the injection should be limited to 750 ul to prevent discomfort to the animals. For large or obese mouse strains, this may require that the concentration of the anesthetic be increased to permit smaller volume injections. The correct level of anesthesia has been reached when the mouse shows no palpebral or tail-pinch reflexes, generally within ~5 min of the injection. A surgical level of anesthesia should persist for 45 min to 1 hr.

Mount mouse to stereotaxic frame

2. Swing the nose clamp and incisor bar down and out of the way, and then lock one of the ear bars at ~3.5 mm from center. Set a heating pad on low (~35C) and place it on the stereotaxic frame to maintain body temperature and raise the animal to the level of the ear bars. While supporting the animal's head from beneath, guide the tip of the locked ear bar into the external auditory meatus and hold firmly. Slide the other ear bar into the opposite ear and apply steadily increasing pressure until small popping sounds are heard; this popping indicates that the ear bars are properly inserted to the tympanic membranes.



The animals will show some hearing loss immediately after the surgery, but hearing seems normal after one week of post-operative recovery. For experiments where normal hearing is critical, non-rupture ear bars may be preferred. However, the head will not be held as stably with this style of ear bar, and the accuracy and precision of the surgery may suffer as a result.

If mounting an adult mouse and the total distance between the ear bars is <5 mm, it is very likely that the ear bars are not in their proper place within the sockets. Also, if gentle lateral pressure moves the snout >2 mm in either direction, ensure that the ear bars are properly seated within the sockets and apply more pressure.

3. Swing the incisor bar up and slide it just into the mouth while holding the jaw open with a pair of blunt forceps. Apply downward pressure to the bridge of the nose while sliding the incisor bar back into the mouth. Stop when the incisors drop into the hole in the bar. Swing the nose clamp into position on the bridge of the nose and tighten firmly. Optionally apply ophthalmic ointment or mineral oil to the eyes to keep them from drying out and protect them from accidental spills.



Perform surgery and injection

4. Disinfect the scalp with Betadine and make an incision along the midline with a scalpel. Using surgical scissors, extend this incision forward to the back of the eyes and backward to between the ears. Use caution to avoid damaging the muscles that insert on the external occipital crest on the back of the skull. Set Agricola-style retractors in the incision to expose the skull, and scrape away the transparent pericranial tissues with a cotton swab.

5. Fit the monoinjector to the stereotaxic frame and take DV measurements at bregma and lambda by lowering the tip of the injection needle until it is just touching these structures. Raise or lower the incisor bar/nose clamp to bring the bregma and lambda DV coordinates within 0.1 mm of each other. Zero the DV scale at bregma (see Fig. 3.10.1). Fit the sighting scope to the stereotaxic frame, align the crosshairs with bregma and zero the AP and ML scales.

6. Fit the drill to the stereotaxic frame, set it at the target AP and ML coordinates and drill a hole through the skull at a rate of ~25 to 50 um/sec.



The diameter of the hold will depend upon the size of the cannula or monoinjector needle used. If a 24-G cannula or monoinjector needle is used, then a diameter of ~0.57 mm works well. As a general rule, the depth of the hole should be ~200 um from the surface of the skull. However, the thickness of the skull varies and, depending on the stereotaxic coordinates being utilized, the depth may have to be adjusted to ensure that the hole is completely through the skull while minimizing damage to the brain.

More accurate injections can be achieved by using a correction factor for determining the target coordinates. To get this correction factor, divide the bregma-to-lambda distance of each animal by the bregma-to-lambda distance from a stereotaxic atlas. Multiply the coordinates given by the atlas by this number to determine the corrected coordinates.

7. Fit the monoinjector to the stereotaxic frame. Lower the needle into the hole to the target DV coordinate and inject. Leave the injection needle in place for at least 30 sec after completion of the injection and before removal of the needle.



Injections directly into brain tissue should not be applied at >0.5 ul/min, however, intracerebroventricular injections can be carried out a faster rate (1 um/min).

8. Remove the monoinjector and seal the hole with bone wax. Suture the incision and remove the animal from the stereotaxic frame. Keep the animal warm during recovery by placing its cage on a heating pad set on low (~35C). House animals individually until the incision has healed to prevent them from chewing each other's stitches.


From Current Protocols in Neuroscience Online


Copyright © 2003 John Wiley & Sons, Inc. All rights reserved.





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