A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

This protocol details the use of a positioning device for the intranasal delivery of biomolecules to the mouse CNS. The use of this device significantly reduces handling of mice and time of treatment resulting in improved efficiencies and reproducibility of the technique. To begin this procedure, assess the anesthesia level of the mice by pedal reflex in order to maintain the surgical plane.

Next, place the positioning device at an appropriate distance and height to allow convenient access to all required reagents. Then, apply ointment on the animal's eyes. Strap one mouse with chair belts, making sure the mouse's forelimbs provide natural support while it is in a relaxed position without any discomfort.

Repeat the procedures for the remaining mice. Gently place each mouse on the designated chair by laying its back parallel to the backs support of the chair and at 90 degrees to the chair seat. Let the animal lie naturally in the head down and forward position without pushing or pressing.

Once all the mice are strapped, immediately start the intranasal inoculation. To perform intranasal delivery, load the micropipette with two microliters of RVG9R siRNA complex solution. Hold the pipette in the dominant hand and support it with the other hand to avoid uncontrolled movements while administering siRNA.

Place about a two microliter droplet very close to one nostril so that the mouse can directly inhale it. If a tiny drop is not easily formed, replace the pipette tip with a new one and repeat the operation. Gently push the mouse head down with index finger for five seconds while it is inhaling and maintain the position.

To prevent drainage of drug into the lungs it is important to sedately push the mouse head down with the index finger for five seconds while the mouse is inhaling. Start a stopwatch to clock three to four minutes after this to time inoculation into the second nare of the first mouse. This time interval is necessary for the mouse to complete inhalation of the first dose and restore normal breathing.

In the three to four minute interval between inoculation of alternate nares on the first mouse, complete intranasal inoculation of one nare in each of the remaining three mice placed on the device. When the stopwatch sounds, inoculate the second nostril on the first mouse and repeat the procedure for the other mice as above. Repeat the procedure until the 20 to 30 microliters is delivered for all four mice.

It will take about 30 to 45 minutes to complete the entire inoculation procedure. Return the animals back to their designated cages. Do not leave the mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.

To assess gene silencing, extract RNA from various brain regions with an RNA purification kit, reverse transcription into cDNA using a cDNA synthesis kit, and perform qPCR with primer pairs. Most brain delivery of the RVG9R peptide labeled with Alexa Fluor 488 was first tested by using the mouse positioning device described here. At 48 hours post inoculation, various organs were excised to measure A488 fluorescence.

Nothing was detected in any of the organs in the negative controls. On the other hand, a strong fluorescent A488 signal was detected exclusively in the brains of the RVG9R inoculated group. In addition, the mouse placement position was compared to the supine position method and the awake method for efficacy.

The results indicated that positioning the mice head down and forward improved the penetrance and deposition of RVG9R A488 throughout the brain tissue. To further confirm siRNA delivery to the brain, full brain scanning was performed after a single intranasal administration of RVG9R complexed to Cy5 labeled siRNA. In contrast to PBS or RVM9R, complexing with RVG9R resulted in a strong accumulation of siRNA in major brain regions including the olfactory bulb, the cortex, the hippocampus, the thalamus, the hypothalamus, the mid-brain, and the cerebellum.

The positioning device can be used for the delivery of drugs or molecules to the mouse CNS. The applications extend to the testing of therapeutics as well as examining cellular mechanisms in the CNS. This positioning device has been used by the investigators for demonstrating the potency of siRNAs in treating West Nile Virus disease, as well as exploring therapies for other CNS diseases.