A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.
Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer’s mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.
1. Acclimation to Handling for Intranasal Dosing to Awake Mice
Introduction
Mice must be acclimated to handling for a period of two-four weeks before the onset of intranasal dosing. Acclimation to handling is important, as it helps ensure a correct body position for maximum effectiveness of awake intranasal drug delivery. In addition, mice that have not been properly accustomed to this process can have a severe anxiety reaction after dosing. Mice should spend about 2-3 days on each of nine steps before progressing to the next step, depending upon the animal’s comfort to handling. The mouse’s stress level should be used as a measure of progress. This means monitoring the mouse’s movements, the amount/frequency of urination, defecation, trembling, and biting. If a mouse has a high stress response, stay on that step before progressing to the next until the response is reduced. A sample acclimation schedule can be seen in Table 1. Acclimation of the mice should progress through the following once-a-day steps. Performing these steps more than once per day, in an attempt to accelerate the acclimation, is not recommended.
2. The Intranasal Grip to Immobilize Awake Mice
3. Intranasal Delivery of Drug to Awake Mice
Introduction
Using the intranasal grip, each mouse is restrained twice and held with their neck parallel to the floor while a volume of 20-30 μl of liquid is administered. This volume is safe and efficacious for small-large mice; we typically use 24 μl, and that is described in this protocol. The awake intranasal method can be used in mice of any age, for example in newborn mice for developmental neuroscience research. In newborn mice, a smaller total volume (8 μl) and droplet size (1 μl) should be used to accommodate the smaller volume of the nasal cavity. Drug is typically delivered to two mice at a time, alternating rest periods to make the most efficient use of time and keep on schedule. Set-up will be a little different depending on if you are dosing group housed mice or individually housed mice. With the individually housed mice, it is helpful to place all of your materials in between the cages to prevent mice from crossing over to the other cage top. With the group-housed mice, dose from that cage only. It is important that each mouse in a group-housed cage receives the same intranasal treatment, as potential for cross contamination of two different treatments exists due to drug expelling into the air or with skin contact. It is highly recommended that a dosing sheet be printed in advance, to remind technicians of treatment assignments, to record any notes of missed drops, to note general health of each animal, etc.
Steps
4. General Animal Handling Techniques for Acclimation and Intranasal Delivery of Drug to Awake Mice
After each acclimation or intranasal dosing session, mice should be given a treat, such as a Cheerio or peeled sunflower seed as positive reinforcement. Treats are given as a reward and to distract them from scratching their noses, potentially causing injury to themselves. When working with group housed mice, enough treats should be dropped for each mouse. Before returning animals to their cages be sure to restore nests to their original states in order to reduce stress. When dosing group housed animals, gloves must be changed between dosing of cages so as not to transfer scent from cage colony to cage colony, as they will become aggressive towards one another.
There are several ways to determine the efficacy of drug delivery. The first is to intranasally administer a dye, such as India ink or methylene blue and to visualize the dye after euthanasia of the animal within the olfactory epithelium and after the brain is removed, entering the CNS through the cribriform plate. The second is to measure the drug delivered either by using a radio-labeled tag or using ELISA detection of the unlabeled molecule. A good example of drug distribution in the CNS after administration to awake mice can be found in Hanson et al. 20127. We expect the olfactory bulb and trigeminal nerve to contain a high concentration of the drug delivered. At 30 min after intranasal administration of 3.65 μmol deferoxamine, concentrations in the trigeminal nerve and olfactory nerve were 29 μM and 13 μM respectively. Drug concentrations will be evident throughout all regions of the brain (in this example a range of 0.6 μM to 4.1 μM was measured) with higher concentrations in areas receiving input from the olfactory and trigeminal nerves. For example, rostral regions of the cortex will contain higher concentrations than caudal cortex and the pons will contain higher concentrations than the midbrain or medulla. In the spinal cord a descending concentration gradient from cervical to lumbar spine is generally observed (in this example a gradient of 1.5 μM to 2.0 μM). Intranasal administration also results in high concentrations of drug reaching the meninges and cervical lymph nodes. The third way to demonstrate efficacy of delivery is to measure a behavioral or biochemical response to the drug administered. For example, intranasal deferoxamine treatment of transgenic Alzheimer’s mice resulted in improved performance in a spatial memory task7 and phosphorylation of glycogen synthase kinase 3 beta9.
Day # | Day | Action |
1 | M | Hold for ~2-3 min |
2 | Tu | Hold for ~2-3 min |
3 | W | Hold and pet ~2-3 min |
4 | Th | Hold and pet ~2-3 min |
5 | F | Lightly pinch or scruff |
6 | M | Lightly pinch or scruff |
7 | Tu | Scruff and lift |
8 | W | Scruff and lift |
9 | Th | Intranasal Grip |
10 | F | Intranasal Grip |
11 | M | Intranasal (IN) Grip and Invert |
12 | Tu | Intranasal (IN) Grip and Invert |
13 | W | IN Grip, Invert, place pipette tip near nose |
14 | Th | IN Grip, Invert, place pipette tip near nose |
15 | F | IN Grip, Invert, deliver 1 round saline to each nare |
16 | M | IN Grip, Invert, deliver 1 round saline to each nare |
17 | Tu | IN Grip, Invert, deliver 2 rounds saline to each nare |
18 | W | IN Grip, Invert, deliver 2 rounds saline to each nare |
Table 1. Sample Schedule for Acclimation to Handling for Intranasal Dosing to Awake Mice.
Time (min:sec) | Mouse # | Action |
0:00 | 1 | 1st set of drops* in left nostril |
0:15 | 2nd set of drops* in right nostril | |
0:30 | Place on cage top | |
1:00 | 2 | 1st set of drops* in left nostril |
1:15 | 2nd set of drops* in right nostril | |
1:30 | Place on cage top | |
2:00 | 1 | 3rd set of drops* in left nostril |
2:15 | 4th set of drops* in right nostril | |
2:30 | Place in cage with treat | |
3:00 | 2 | 3rd set of drops* in left nostril |
3:15 | 4th set of drops* in right nostril | |
3:30 | Place in cage with treat |
*The pipettor is loaded once with 6 μl, but administered to the mouse as 2 x 3 μl drops.
Table 2. Sample Timeline for Dosing One or Two Mice.
Figure 1. Photos of different stages and angles of holding a mouse for awake intranasal delivery of drug. 1A) Hold the mouse down firmly on the cage top with the dominant hand leaving the shoulders exposed to grip the skin. 1B) Use the thumb and middle finger of the non-dominant hand to grip and pull up skin just behind the shoulder blades. 1C) Use the index finger to pull skin back from between the eyes towards the thumb and middle finger (front view). 1D) Same as 1C (side view). 1E) Hold the mouse upside down using the ring finger and/or pinky to secure the base of the tail to the palm of the hand. The neck of mouse should be horizontal. 1F) Same as E, from other side. 1G) Administer drug with a 20 μl pipettor and gel loading pipette tip. 1H) Same as 1G, close-up view.
Figure 2. A photo of a complete dosing station for 2 individually housed mice. Included are the two mouse cages with enough distance between to keep the mice separate, a pipettor with tips, a container for used tips, a timer, dosing sheet, a post-dosing treat (like sunflower seeds or cheerios), sanitary wipes, a P20 (or P10) pipettor, and the drug to be delivered.
This method shows how mice can be given regular doses of intranasal drug to target the brain without the use of anesthesia. Intranasal drug can be delivered to large groups of mice for extended periods of time with minimal effort. A single group of up to 20 mice can be intranasally treated by a single person in less than one hour. A vast array of molecules, ranging from small iron chelators to growth factors to mesenchymal stem cells, have been demonstrated to rapidly target the CNS and have shown efficacy in rodents models. Proposed mechanisms of direct delivery from the nose to the brain include extracellular transport along the olfactory and trigeminal nerves and bulk flow within the perivascular spaces1,10. Intranasal delivery to the CNS is an especially useful technique because not only is it rapid, but it targets the CNS reducing systemic exposure. For example, intranasal administration of deferoxamine to rats resulted in 3-19 times higher concentrations in brain regions with 28 times lower concentrations in the blood11. In another example, intranasally administered insulin improves memory in Alzheimer’s patients within 15 min without changing the plasma levels of insulin or glucose12. An additional advantage is the non-invasiveness of the intranasal delivery technique.
There are several limitations to this method. First, drug delivery is limited to about 20-30 μl in a single administration, so solubility of the therapeutic will play a role in determining the dose limits. Also, the drug formulation is important as factors like pH and salinity can affect the delivery. Large deviations from natural conditions, such as extreme pH and salinity, can damage the respiratory and olfactory epithelia of mice.13,14 Further, intranasal dosing of unanesthetized mice can be difficult to master, and can take several weeks of training for intranasal “dosers” to be able to minimize “head-wiggle room” and achieve optimal intranasal dosing of drug. With incorrect dosing technique, the drug can drain into the lung or the stomach, which can introduce variability in drug reaching the brain. Signs that this has occurred are breathing difficulties or visualization of liquid in the mouth after administration. If possible, it is advisable to limit new intranasal “dosers” to delivering saline/vehicle to control groups for several weeks or months before they are approved to dose drug by expert trainers. Finally, this awake intranasal method is limited to mice, as awake rats and larger animals are not as easily gripped, and can be overly stressed when held upside down.
The authors have nothing to disclose.
The authors would like to thank the HealthPartners Institutional Animal Care and Use Committee at Regions Hospital.
Materials | Company | Cat # | Comment |
P20 pipettor set at 6 μl | |||
Gel loading pipette tips | |||
Timer | |||
Waste receptacle for tips | |||
Dosing sheet for notes | |||
Pen | |||
Drug and vehicle | |||
Container to hold drug vials | |||
Animals to be dosed | |||
Spare gloves | |||
Paper towels to wipe urine | |||
Table 3. Materials Needed |