In Vivo Adenovirus-Mediated Gene Transfer into the CNS of Adult Rats Protocol

Experiment Summary

Viral vector-mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of neurobiological research and for gene therapy of neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose. Adenoviruses are easily purified to the high titers required for in vivo administration, and they are efficient in transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression. This protocol describes a procedure for the direct injection of adenovirus into the CNS using stereotaxic guidance. In the protocol below, the technique is adapted to gaseous anesthesia and viral vector injection.

Materials

Adenoviral vector
Sterile phosphate-buffered saline (PBS; APPENDIX 2A), pH 7.4
Adult rat (250 g body weight; rats 200 to 350 g may be fitted into the frame; however the stereotaxic coordinates may not be accurate with larger or smaller animals, since standard atlases illustrate the brain of 250-g rats)
70% (v/v) ethanol/isopropyl alcohol
Stereomicroscope
Rubber balloon
Stereotaxic frame with rat adapter and blunt ear bars (Stoelting), modified for gas anesthesia
Gas anesthetic trolley with the following components: Halothane gas anesthetic, Halothane vaporizer (e.g., Fluotec), Medical oxygen cylinder, Medical nitrous oxide cylinder, Induction chamber, Halothane scavenger (e.g., Fluovac)
Electric drill with 1.75-mm drill bit
Heat pad
Surgical shavers (Stoelting)
Scalpel and blades
Skin retractors
Cotton swabs
10-µl Hamilton syringe with needle (model 701RN, Fisher)
Fiber optic illuminator with twin goose-neck pipes (Leica)
Sterile 23-G or 25-G hypodermic needles
1-ml syringes
Curved and straight forceps
Holding scissors
Sharp scissors
Sterile gauze
Chromic catgut absorbable sutures

Fig. 1 Stereotaxic frame modified for inhalational anesthesia.

Procedure

Position the stereotaxic frame relative to the light microscope and light box such that the microscope is focused on the ear bars of the frame. Stretch a rubber balloon over the mouthpiece of the stereotaxic unit and cut a small hole in the end to allow insertion of the animal’s nose. Position the anesthetic trolley such that the anesthetic tubing can be connected to the stereotaxic frame, and lay out the drill, heat pad, and sterilized surgical tools next to the frame.
Dilute the adenovirus preparation with sterile saline solution (pH 7.4) or PBS (pH 7.4) such that the required number of infectious units can be administered in the appropriate volume (see Critical Parameters). Store on ice in a microcentrifuge tube until required.
Place the animal in the induction chamber and anesthetize with 4% halothane gas, vaporized with an oxygen/nitrous oxide mix (66% oxygen:33% nitrous oxide, i.e., O₂ set at a flow rate of 1500 ml/min and nitrous oxide at a flow rate of 750 ml/min).
When the animal is fully anesthetized, route the flow of halothane to the stereotaxic unit, remove the rat from the induction chamber, and quickly shave the fur on top of the head. Open the mouth of the rat and fit the mouthpiece of the stereotaxic frame such that the animal’s nose is entirely enclosed within the rubber balloon mask. Reduce the halothane level to 1.5% of the carrier gas.
Slide the ear bars into each ear canal and tighten in place so that the head of the animal is firmly positioned and does not wobble. Check that the top of the head is lying horizontally. Place the heat pad underneath the animal to prevent hypothermia during surgical anesthesia.
When the animal is positioned firmly and correctly within the frame, place a drop of sterile saline solution into each of the eyes and swab the surgical site with a cotton swab dipped in 70% ethanol/isopropyl alcohol.
Ensure that the animal is fully anesthetized by checking the lack of responses to footpad and tail pinching. Using a scalpel, make a midline incision into the skin, from above the eyes to the level of the ears.
Use skin retractors to hold back the skin on either side of the incision.
Remove the connective tissue covering the top of the skull by cleaning the cranium with a cotton swab.
Load the Hamilton syringe with the adenovirus solution and expel a small amount of the virus onto a cotton swab to verify the needle is not blocked. Clamp the needle into position on the frame.
Direct the light beams from the fiberoptic light pipes onto the exposed skull and focus the microscope onto bregma.
Position the syringe over the skull and tighten into place. While viewing the brain through the microscope, position the needle using the 3 slide rules so that the needle bevel is directly over bregma.
Read the coordinates of bregma from the frame. Calculate the new coordinates of the site of injection by adding or subtracting the appropriate lateral and anterior/posterior values from bregma. Move the needle to these new coordinates.
Lower the needle at the new coordinates, so that it is just touching the surface of the skull (be careful not to damage the needle point), and mark this position with a small dot using a very fine marker pen. Raise the needle to allow drilling.
Viewing the surface of the skull through the microscope, drill a small hole approximately 2 mm in diameter most of the way through the skull at the position marked by the small dot.
Stop drilling when the base of the hole becomes translucent. Perforate the remaining thin layer of skull with a sterile needle and using a pair of sharp, curved forceps, carefully remove this remaining layer of bone to expose the dura matter.
Using a sterile, bent needle, carefully perforate the dura and remove as much of the membrane as needed to expose the surface of the brain.
Lower the Hamilton needle into the hole until it just touches the surface of the brain and read the vertical coordinate of this position. Calculate the new vertical coordinate of the site of injection.
Lower the needle into the brain to the site of injection and wait 2 to 3 min before slowly depressing the syringe plunger by 0.5 µl, over a further minute. Wait 1 min for the virus solution to infuse into the brain. Inject a further 0.5 µl of virus and wait a further minute. Repeat until the entire 2 µl of virus has been administered. Wait for 5 min after the final administration.
Remove the needle from the brain very slowly and close the skin incision with sutures.
Swab the sutured area with sterile saline, turn off the anesthetic and nitrous oxide while maintaining oxygen flow to the unit, and allow the animal to recover.

* For research use only. Not intended for any clinical use.

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In Vivo Adenovirus-Mediated Gene Transfer into the CNS of Adult Rats Protocol

Experiment Summary

Materials

Procedure

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